Mammalian apoptosis inhibitor protein gene family, primers, probes and detection methods

ABSTRACT

Disclosed is substantially pure DNA encoding mammalian IAP polypeptides; substantially pure polypeptides; and methods of using such DNA to express the IAP polypeptides in cells and animals to inhibit apoptosis. Also disclosed are conserved regions characteristic of the IAP family and primers and probes for the identification and isolation of additional IAP genes. In addition, methods for treating diseases and disorders involving apoptosis are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National phase application of PCT IB96/01022,and claims priority to, and is a continuation-in-part of U.S. Ser. No.08/576,956, filed Dec. 22, 1995 now U.S. Pat. No. 6,156,535, which is acontinuation-in-part of U.S. Ser. No. 08/511,485, filed Aug. 4, 1995,now issued as U.S. Pat. No. 5,919,912.

BACKGROUND OF THE INVENTION

The invention relates to apoptosis.

There are two general ways by which cells die. The most easilyrecognized way is by necrosis, which is usually caused by an injury thatis severe enough to disrupt cellular homeostasis. Typically, the cell'sosmotic pressure is disturbed and, consequently, the cell swells andthen ruptures. When the cellular contents are spilled into thesurrounding tissue space, an inflammatory response often ensues.

The second general way by which cells die is referred to as apoptosis,or programmed cell death. Apoptosis often occurs so rapidly that it isdifficult to detect. This may help to explain why the involvement ofapoptosis in a wide spectrum of biological processes has only recentlybeen recognized.

The apoptosis pathway has been highly conserved throughout evolution,and plays a critical role in embryonic development, viral pathogenesis,cancer, autoimmune disorders, and neurodegenerative disease. Forexample, inappropriate apoptosis may cause or contribute to AIPS,Alzheimer's Disease, Parkinson's Disease, Amyotrophic Lateral Sclerosis(ALS), retinitis pigmentosa and other diseases of the retina,myelodysplastic syndrome (e.g. aplastic anemia), toxin-induced liverdisease, including alcoholism, and ischemic injury (e.g. myocardialinfarction, stroke, and reperfusion injury). Conversely, the failure ofan apoptotic response has been implicated in the development of cancer,particularly follicular lymphoma, p53-mediated carcinomas, andhormone-dependent tumors, in autoimmune disorders, such as lupuserythematosis and multiple sclerosis, and in viral infections, includingthose associated with herpes virus, poxvirus, and adenovirus.

In patients infected with HIV-1, mature CD4⁺ T lymphocytes respond tostimulation from mitogens or super-antigens by undergoing apoptosis.However, the great majority of these cells are not infected with thevirus. Thus, inappropriate antigen-induced apoptosis could beresponsible for the destruction of this vital part of the immune systemin the early stages of HIV infection.

Baculoviruses encode proteins that are termed inhibitors of apoptosisproteins (IAPs) because they inhibit the apoptosis that would otherwiseoccur when insect cells are infected by the virus. These proteins is arethought to work in a manner that is independent of other viral-proteins.The baculovirus IAP genes include sequences encoding a ring zincfinger-like motif (RZF), which is presumed to be directly involved inDNA binding, and two N-terminal domains that consist of a 70 amino acidrepeat motif termed a BIR domain (Baculovirus IAP Repeat).

SUMMARY OF THE INVENTION

In general, the invention features a substantially pure DNA molecule,such as a genomic, cDNA, or synthetic DNA molecule, that encodes amammalian IAP polypeptide. This DNA may be incorporated into a vector,into a cell, which may be a mammalian, yeast, or bacterial cell, or intoa transgenic animal or embryo thereof. In preferred embodiments, the DNAmolecule is a murine gene (e.g., m-xiap, m-hiap-1, or m-hiap-2) or ahuman gene (e.g., xiap, hiap-1, or hiap-2). In most preferredembodiments the IAP gene is a human IAP gene. In other various preferredembodiments, the cell is a transformed cell. In related aspects, theinvention features a transgenic animal containing a transgene thatencodes an IAP polypeptide that is expressed in or delivered to tissuenormally susceptible to apoptosis, i.e., to a tissue that may be harmedby either the induction or repression of apoptosis. In yet anotheraspect, the invention features DNA encoding fragments of IAPpolypeptides including the BIR domains and the RZF domains providedherein.

In specific embodiments, the invention features DNA sequencessubstantially identical to the DNA sequences shown in FIGS. 1-6, orfragments thereof. In another aspect, the invention also features RNAwhich is encoded by the DNA described herein. Preferably, the RNA ismRNA. In another embodiment the RNA is antisense RNA.

In another aspect, the invention features a substantially purepolypeptide having a sequence substantially identical to one of the IAPamino acid sequences shown in FIGS. 1-6.

In a second aspect, the invention features a substantially pure DNAwhich includes a promoter capable of expressing the IAP gene in a cellsusceptible to apoptosis. In preferred embodiments, the IAP gene isxiap, hiap-1, or hiap-2. Most preferably, the genes are human or mousegenes. The gene encoding hiap-2 may be the full-length gene, as shown inFIGS. 3A-3G, or a truncated variant, such as a variant having a deletionof the sequence boxed in FIG. 3E.

In preferred embodiments, the promoter is the promoter native to an IAPgene. Additionally, transcriptional and translational regulatory regionsare, preferably, those native to an IAP gene. In another aspect, theinvention provides transgenic cell lines and transgenic animals. Thetransgenic cells of the invention are preferably cells that are alteredin their apoptotic response. In preferred embodiments, the transgeniccell is a fibroblast, neuronal cell, a lymphocyte cell, a glial cell, anembryonic stem cell, or an insect cell. Most preferably, the neuron is amotor neuron and the lymphocyte is a CD4⁺ T cell.

In another aspect, the invention features a method of inhibitingapoptosis that involves producing a transgenic cell having a transgeneencoding an IAP polypeptide. The transgene is integrated into the genomeof the cell in a way that allows for expression. Furthermore, the levelof expression in the cell is sufficient to inhibit apoptosis.

In a related aspect, the invention features a transgenic animal,preferably a mammal, more preferably a rodent, and most preferably amouse, having either increased copies of at least one IAP gene insertedinto the genome (mutant or wild-type), or a knockout of at least one IAPgene in the genome. The transgenic animals will express either anincreased or a decreased amount of IAP polypeptide, depending on theconstruct used and the nature of the genomic alteration. For example,utilizing a nucleic acid molecule that encodes all or part of an IAP toengineer a knockout mutation in an IAP gene would generate an animalwith decreased expression of either all or part of the corresponding IAPpolypeptide. In contrast, inserting exogenous copies of all or part ofan IAP gene into the genome, preferably under the control of activeregulatory and promoter elements, would lead to increased expression orthe corresponding IAP polypeptide.

In another aspect, the invention features a method of detecting an IAPgene in a cell by contacting the IAP gene, or a portion thereof (whichis greater than 9 nucleotides, and preferably greater than 18nucleotides in length), with a preparation of genomic DNA from the cell.The IAP gene and the genomic DNA are brought into contact underconditions that allow for hybridization (and therefore, detection) ofDNA sequences in the cell that are at least 50% identical to the DNAencoding HIAP-1, HIAP-2, or XIAP polypeptides.

In another aspect, the invention features a method of producing an IAPpolypeptide. This method involves providing a cell with DNA encoding allor part of an IAP polypeptide (which is positioned for expression in thecell), culturing the cell under conditions that allow for expression ofthe DNA, and isolating the IAP polypeptide. In preferred embodiments,the IAP polypeptide is expressed by DNA that is under the control of aconstitutive or inducible promotor. As described herein, the promotormay be a heterologous promotor.

In another aspect, the invention features substantially pure mammalianIAP polypeptide. Preferably, the polypeptide includes an amino acidsequence that is substantially identical to all, or to a fragment of,the amino acid sequence shown in any one of FIGS. 1-4. Most preferably,the polypeptide is the XIAP, HIAP-1, HIAP-2, M-XIAP, M-HIAP-1, orM-HIAP-2 polypeptide. Fragments including one or more BIR domains (tothe exclusion of the RZF), the RZF domain (to the exclusion of the BIRdomains), and a RZF domain with at least one BIR domain, as providedherein, are also a part of the invention.

In another aspect, the invention features a recombinant mammalianpolypeptide that is capable of modulating apoptosis. The polypeptide mayinclude at least a ring zinc finger domain and a BIR domain as definedherein. In preferred embodiments, the invention features (a) asubstantially pure polypeptide, and (b) an oligonucleotide encoding thepolypeptide. In instances were the polypeptide includes a ring zincfinger domain, the ring zinc finger domain will have a sequenceconforming to:Glu-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa2-Xaa1-Xaa1-Xaa1-Cys-Lys-Xaa3-Cys-Met-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa3-Xaa1-Phe-Xaa1-Pro-Cys-Gly-His-Xaa1-Xaa1-Xaa1-Cys-Xaa1-Xaa1-Cys-Ala-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Cys-Pro-Xaa1-Cys,where Xaa1 is any amino acid, Xaa2 is Glu or Asp, Xaa3 is Val or Ile(SEQ ID NO:1); and where the polypeptide includes at least one BIRdomain, the BIR domain will have a sequence conforming to:Xaa1-Xaa1-Xaa1-Arg-Leu-Xaa1-Thr-Phe-Xaa1-Xaa1-Trp-Pro-Xaa2-Xaa1-Xaa1-Xaa2-Xaa2-Xaa1-Xaa1-Xaa1-Xaa1-Leu-Ala-Xaa1-Ala-Gly-Phe-Tyr-Tyr-Xaa1-Gly-Xaa1-Xaa1-Asp-Xaa1-Val-Xaa1-Cys-Phe-Xaa1-Cys-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Trp-Xaa1-Xaa1-Xaa1-Asp-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-His-Xaa1-Xaa1-Xaa1-Xaa1-Pro-Xaa1-Cys-Xaa1-Phe-Val,where Xaa1may be any amino acid and Xaa2 may be any amino acid or may beabsent (SEQ ID NO:2).

In various preferred embodiments the polypeptide has at least two or,more preferably at least three BIR domains, the RZF domain has one ofthe IAP sequences shown in FIGS. 6A-6F, and the BIR domains arecomprised of BIR domains shown in FIGS. 5A-5F. In other preferredembodiments the BIR domains are at the amino terminal end of the proteinrelative to the RZF domain, which is at or near the carboxyl terminus ofthe polypeptide.

In another aspect, the invention features an IAP gene isolated accordingto the method involving: (a) providing a sample of DNA; (b) providing apair of oligonucleotides having sequence homology to a conserved regionof an IAP disease-resistance gene; (c) combining the pair ofoligonucleotides with the cell DNA sample under conditions suitable forpolymerase chain reaction-mediated DNA amplification; and (d) isolatingthe amplified IAP gene or fragment thereof.

In preferred embodiments, the amplification is carried out using areverse-transcription polymerase chain reaction, for example, the RACEmethod. In another aspect, the invention features an IAP gene isolatedaccording to the method involving: (a) providing a preparation of DNA;(b) providing a detectably labelled DNA sequence having homology to aconserved region of an IAP gene; (c) contacting the preparation of DNAwith the detectably-labelled DNA sequence under hybridization conditionsproviding detection of genes having 50% or greater nucleotide sequenceidentity; and (d) identifying an IAP gene by its association with thedetectable label.

In another aspect, the invention features an IAP gene isolated accordingto the method involving: (a) providing a cell sample; (b) introducing bytransformation into the cell sample a candidate IAP gene; (c) expressingthe candidate IAP gene within the cell sample; and (d) determiningwhether the cell sample exhibits an altered apoptotic response, wherebya response identifies an IAP gene.

In another aspect, the invention features a method of identifying an IAPgene in a cell, involving: (a) providing a preparation of cellular DNA(for example, from the human genome or a cDNA library (such as a cDNAlibrary isolated from a cell type which undergoes apoptosis); (b)providing a detectably-labelled DNA sequence (for example, prepared bythe methods of the invention) having homology to a conserved region ofan IAP gene; (c) contacting the preparation of cellular DNA with thedetectably-labelled DNA sequence under hybridization conditionsproviding detection of genes having 50% nucleotide or greater sequenceidentity; and (d) identifying an IAP gene by its association with thedetectable label.

In another aspect, the invention features a method of isolating an IAPgene from a recombinant library, involving: (a) providing a recombinantlibrary; (b) contacting the library with a detectably-labelled genefragment produced according to the PCR method of the invention underhybridization conditions providing detection of genes having 50% orgreater nucleotide sequence identity; and (c) isolating an IAP gene byits association with the detectable label. In another aspect, theinvention features a method of identifying an IAP gene involving: (a)providing a cell tissue sample; (b) introducing by transformation intothe cell sample a candidate IAP gene; (c) expressing the candidate IAPgene within the cell sample; and (d) determining whether the cell sampleexhibits inhibition of apoptosis, whereby a change in (i.e. modulationof) apoptosis identifies an IAP gene. Preferably, the cell sample is acell type that may be assayed for apoptosis (e.g., T cells, B cells,neuronal cells, baculovirus-infected insect cells, glial cells,embryonic stem cells, and fibroblasts). The candidate IAP gene isobtained, for example, from a cDNA expression library, and the responseassayed is the inhibition of apoptosis.

In another aspect, the invention features a method of inhibitingapoptosis in a mammal wherein the method includes: (a) providing DNAencoding at least one IAP polypeptide to a cell that is susceptible toapoptosis; wherein the DNA is integrated into the genome of the cell andis positioned for expression in the cell; and the IAP gene is under thecontrol of regulatory sequences suitable for controlled expression ofthe gene(s); wherein the IAP transgene is expressed at a levelsufficient to inhibit apoptosis relative to a cell lacking the IAPtransgene. The DNA integrated into the genome may encode all or part ofan IAP polypeptide. It may, for example, encode a ring zinc finger andone or more BIR domains. In contrast, it may encode either the ring zincfinger alone, or one or more BIR domains alone. Skilled artisans willappreciate that IAP polypeptides may also be administered directly toinhibit undesirable apoptosis.

In a related aspect, the invention features a method of inhibitingapoptosis by producing a cell that has integrated, into its genome, atransgene that includes the IAP gene, or a fragment thereof. The IAPgene may be placed under the control of a promoter providingconstitutive expression of the IAP gene. Alternatively, the IAPtransgene may be placed under the control of a promoter that allowsexpression of the gene to be regulated by environmental stimuli. Forexample, the IAP gene may be expressed using a tissue-specific or celltype-specific promoter, or by a promoter that is activated by theintroduction of an external signal or agent, such as a chemical signalor agent. In preferred embodiments the cell is a lymphocyte, a neuronalcell, a glial cell, or a fibroblast. In other embodiments, the cell inan HIV-infected human, or in a mammal suffering from a neurodegenerativedisease, an ischemic injury, a toxin-induced liver disease, or amyelodysplastic syndrome.

In a related aspect, the invention provides a method of inhibitingapoptosis in a mammal by providing an apoptosis-inhibiting amount of IAPpolypeptide. The IAP polypeptide may be a full-length polypeptide, or itmay be one of the fragments described herein.

In another aspect, the invention features a purified antibody that bindsspecifically to an IAP family protein. Such an antibody may be used inany standard immunodetection method for the identification of an IAPpolypeptide. Preferably, the antibody binds specifically to XIAP,HIAP-1, or HIAP-2. In various embodiments, the antibody may react withother IAP polypeptides or may be specific for one or a few IAPpolypeptides. The antibody may be a monoclonal or a polyclonal antibody.Preferably, the antibody reacts specifically with only one of the IAPpolypeptides, for example, reacts with murine and human xiap, but notwith hiap-1 or hiap-2 from other mammalian species.

The antibodies of the invention may be prepared by a variety of methods.For example, the IAP polypeptide, or antigenic fragments thereof, can beadministered to an animal in order to induce the production ofpolyclonal antibodies. Alternatively, antibodies used as describedherein may be monoclonal antibodies, which are prepared using hybridomatechnology (see, e.g., Kohler et al., Nature 256:495, 1975; Kohler etal., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol.6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T CellHybridomas, Elsevier, N.Y., 1981). The invention features antibodiesthat specifically bind human or murine IAP polypeptides, or fragmentsthereof. In particular the invention features “neutralizing” antibodies.By “neutralizing” antibodies is meant antibodies that interfere with anyof the biological activities of IAP polypeptides, particularly theability of IAPs to inhibit apoptosis. The neutralizing antibody mayreduce the ability of IAP polypeptides to inhibit polypeptides by,preferably 50%, more perferably by 70, and most preferably by 90% ormore. Any standard assay of apoptosis, including those described herein,may be used to assess neutralizing antibodies.

In addition to intact monoclonal and polyclonal anti-IAP antibodies, theinvention features various genetically engineered antibodies, humanizedantibodies, and antibody fragments, including F(ab′)2, Fab′, Fab, Fv andsFv fragments. Antibodies can be humanized by methods known in the art,e.g., monoclonal antibodies with a desired binding specificity can becommercially humanized (Scotgene, Scotland; Oxford Molecular, Palo

Alto, Calif.). Fully human antibodies, such as those expressed intransgenic animals, are also features of the invention (Green et al.,Nature Genetics 7:13-21, 1994).

Ladner (U.S. Pat. Nos. 4,946,778 and 4,704,692) describes methods forpreparing single polypeptide chain antibodies. Ward et al. (Nature341:544-546, 1989) describe the preparation of heavy chain variabledomains, which they term “single domain antibodies,” which have highantigen-binding affinities. McCafferty et al. (Nature 348:552-554, 1990)show that complete antibody V domains can be displayed on the surface offd bacteriophage, that the phage bind specifically to antigen, and thatrare phage (one in a million) can be isolated after affinitychromatography. Boss et al. (U.S. Pat. No. 4,816,397) describe variousmethods for producing immunoglobulines, and immunologically functionalfragments thereof, which include at least the variable domains of theheavy and light chain in a single host cell. Cabilly et al. (U.S. Pat.No. 4,816,567) describe methods for preparing chimeric antibodies.

In another aspect, the invention features a method of identifying acompound that modulates apoptosis. The method includes providing a cellexpressing an IAP polypeptide, contacting the cell with a candidatecompound, and monitoring the expression of an IAP gene. An alteration inthe level of expression of the IAP gene indicates the presence of acompound which modulates apoptosis. The compound may be an inhibitor oran enhancer of apoptosis. In various preferred embodiments, the cell isa fibroblast, a neuronal cell, a glial cell, a lymphocyte (T cell or Bcell), or an insect cell; the polypeptide expression being monitored isXIAP, HIAP-1, HIAP-2, M-XIAP, M-HIAP-1, or M-HIAP-2 (i.e., human ormurine).

In a related aspect, the invention features methods of detectingcompounds that modulate apoptosis using the interaction trap technologyand IAP polypeptides, or fragments thereof, as a component of the bait.In preferred embodiments, the compound being tested as a modulator ofapoptosis is also a polypeptide.

In another aspect, the invention features a method for diagnosing a cellproliferation disease, or an increased likelihood of such a disease,using an IAP nucleic acid probe or antibody. Preferably, the disease isa cancer. Most preferably, the disease is selected from the groupconsisting of promyelocytic leukemia, a HeLa-type carcinoma, chronicmyelogenous leukemia (preferably using xiap or hiap-2 related probes),lymphoblastic leukemia (preferably using a xiap related probe),Burkitt's lymphoma (preferably using an hiap-1 related probe),colorectal adenocarcinoma, lung carcinoma, and melanoma (preferablyusing a xiap probe). Preferably, a diagnosis is indicated by a 2-foldincrease in expression or activity, more preferably, at least a 10-foldincrease in expression or activity.

Skilled artisans will recognize that a mammalian IAP, or a fragmentthereof (as described herein), may serve as an active ingredient in atherapeutic composition. This composition, depending on the IAP orfragment included, may be used to modulate apoptosis and thereby treatany condition that is caused by a disturbance in apoptosis.

In addition, apoptosis may be induced in a cell by administering to thecell a negative regulator of the IAP-dependent anti-apoptotic pathway.The negative regulator may be, but is not limited to, an IAP polypeptidethat includes a ring zinc finger, and an IAP polypeptide that includes aring zinc finger and lacks at least one BIR domain. Alternatively,apoptosis may be induced in the cell by administering a gene encoding anIAP polypeptide, such as these two polypeptides. In yet another method,the negative regulator may be a purified antibody, or a fragmentthereof, that binds specifically to an IAP polypeptide. For example, theantibody may bind to an approximately 26 kDa cleavage product of an IAPpolypeptide that includes at least one BIR domain but lacks a ring zincfinger domain. The negative regulator may also be an IAP antisense mRNAmolecule.

As summarized above, an IAP nucleic acid, or an IAP polypeptide may beused to modulate apoptosis. Furthermore, an IAP nucleic acid, or an IAPpolypeptide, may be used in the manufacture of a medicament for themodulation of apoptosis.

By “IAP gene” is meant a gene encoding a polypeptide having at least oneBIR domain and a ring zinc finger domain which is capable of modulating(inhibiting or enhancing) apoptosis in a cell or tissue when provided byother intracellular or extracellular delivery methods. In preferredembodiments the IAP gene is a gene having about 50% or greaternucleotide sequence identity to at least one of the IAP amino acidencoding sequences of FIGS. 1-4 or portions thereof. Preferably, theregion of sequence over which identity is measured is a region encodingat least one BIR domain and a ring zinc finger domain. Mammalian IAPgenes include nucleotide sequences isolated from any mammalian source.Preferably, the mammal is a human.

The term “IAP gene” is meant to encompass any member of the family ofapoptosis inhibitory genes, which are characterized by their ability tomodulate apoptosis. An IAP gene may encode a polypeptide that has atleast 20%, preferably at least 30%, and most preferably at least 50%amino acid sequence identity with at least one of the conserved regionsof one of the IAP members described herein (i.e., either the BIR or ringzinc finger domains from the human or murine xiap, hiap-1 and hiap-2).Representative members of the IAP gene family include, withoutlimitation, the human and murine xiap, hiap-1, and hiap-2 genes.

By “IAP protein” or “IAP polypeptide” is meant a polypeptide, orfragment thereof, encoded by an IAP gene.

By “BIR domain” is meant a domain having the amino acid sequence of theconsensus sequence:Xaa1-Xaa1-Xaa1-Arg-Leu-Xaa1-Thr-Phe-Xaa1-Xaa1-Trp-Pro-Xaa2-Xaa1-Xaa1-Xaa2-Xaa2-Xaa1-Xaa1-Xaa1-Xaa1-Leu-Ala-Xaa1-Ala-Gly-Phe-Tyr-Tyr-Xaa1-Gly-Xaa1-Xaa1-Asp-Xaa1-Val-Xaa1-Cys-Phe-Xaa1-Cys-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Trp-Xaa1-Xaa1-Xaa1-Asp-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-His-Xaa1-Xaa1-Xaa1-Xaa1-Pro-Xaa1-Cys-Xaa1-Phe-Val,wherein Xaa1is any amino acid and Xaa2 is any amino acid or is absent(SEQ ID NO:2). Preferably, the sequence is substantially identical toone of the BIR domain sequences provided for xiap, hiap-1, hiap-2herein.

By “BIR domain” is also meant a domain having the amino acid sequance ofamino acids 26-93, 163-230, or 265-330 of XIAP (SEQ ID NO: 4), aminoacids 26-93, 1163-230, or 264-329 of M-XIAP (SEQ ID NO: 6), amino acids29-96, 169-235, or 255-322 of HIAP-1 (SEQ ID NO; 10), amino acids 29-96,169-235, or 255-322 of M-HIAP-1 (SEQ ID NO: 40), amino acids 46-113,184250, 269-336 of HIAP-2 (SEQ ID NO: 8), or amino acids 25-92, 156-222,or 241-308 of M-HIAP-2 (SEQ ID NO: 42).

By “ring zinc finger” or “RZF” is meant a domain having the amino acidsequence of the consensus sequence:Glu-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa2-Xaa1-Xaa1-Xaa1-Cys-Lys-Xaa3-Cys-Met-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa3-Xaa1-Phe-Xaa1-Pro-Cys-Gly-His-Xaa1-Xaa1-Xaa1-Cys-Xaa1-Xaa1-Cys-Ala-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Cys-Pro-Xaa1-Cys,wherein Xaa1is any amino acid, Xaa2 is Glu or Asp, and Xaa3 is Val orIle (SEQ ID NO:1).

Preferably, the sequence is substantially identical to the RZF domainsprovided herein for the human or murine xiap, hiap-1, or hiap-2.

By “ring zinc finger” or “ZF” is also meant a domain having the aminoacid sequence of consisting of amino acids 439-484 of XIAP (SEQ ID NO:4), amino acids 438-483 of M-XIAP (SEQ ID NO: 6), amino acids 546-591 ofHIAP-1 (SEQ ID NO: 10), amino acids 544-589 of M-HIAP-1 (SEQ ID) NO:40), amino acids 560-605 of HIAP-2 (SEQ ID NO: 8), or amino acids or541-578 of M-HIAP-2 (SEQ NO: 42), wherein said polypeptide is capable ofmodulating apoptosis of a mammalia cell.

By “modulating apoptosis” or “altering apoptosis” is meant increasing ordecreasing the number of cells that would otherwise undergo apoptosis ina given cell population. Preferably, the cell population is selectedfrom a group including T cells, neuronal cells, fibroblasts, or anyother cell line known to undergo apoptosis in a laboratory setting(e.g., the baculovirus infected insect cells). It will be appreciatedthat the degree of modulation provided by an IAP or modulating compoundin a given assay will vary, but that one skilled in the art candetermine the statistically significant change in the level of apoptosiswhich identifies an IAP or a compound which modulates an IAP.

By “inhibiting apoptosis” is meant any decrease in the number of cellswhich undergo apoptosis relative to an untreated control. Preferably,the decrease is at least 25%, more preferably the decrease is 50%, andmost preferably the decrease is at least one-fold.

By “polypeptide” is meant any chain of more than two amino acids,regardless of post-translational modification such as glycosylation orphosphorylation.

By “substantially identical” is meant a polypeptide or nucleic acidexhibiting at least 50%, preferably 85%, more preferably 90%, and mostpreferably 95% homology to a reference amino acid or nucleic acidsequence. For polypeptides, the length of comparison sequences willgenerally be at least 16 amino acids, preferably at least 20 aminoacids, more preferably at least 25 amino acids, and most preferably 35amino acids. For nucleic acids, the length of comparison sequences willgenerally be at least 50 nucleotides, preferably at least 60nucleotides, more preferably at least 75 nucleotides, and mostpreferably 110 nucleotides.

Sequence identity is typically measured using sequence analysis softwarewith the default parameters specified therein (e.g., Sequence AnalysisSoftware Package of the Genetics Computer Group, University of WisconsinBiotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Thissoftware program matches similar sequences by assigning degrees ofhomology to various substitutions, deletions, and other modifications.Conservative substitutions typically include substitutions within thefollowing groups: glycine, alanine, valine, isoleucine, leucine;aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine.

By “substantially pure polypeptide” is meant a polypeptide that has beenseparated from the components that naturally accompany it. Typically,the polypeptide is substantially pure when it is at least 60%, byweight, free from the proteins and naturally-occurring organic moleculeswith which it is naturally associated. Preferably, the polypeptide is anIAP polypeptide that is at least 75%, more preferably at least 90%, andmost preferably at least 99%, by weight, pure. A substantially pure IAPpolypeptide may be obtained, for example, by extraction from a naturalsource (e.g. a fibroblast, neuronal cell, or lymphocyte) by expressionof a recombinant nucleic acid encoding an IAP polypeptide, or bychemically synthesizing the protein. Purity can be measured by anyappropriate method, e.g., by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

A protein is substantially free of naturally associated components whenit is separated from those contaminants which accompany it in itsnatural state. Thus, a protein which is chemically synthesized orproduced in acellular system different from the cell from which itnaturally originates will be substantially free from its naturallyassociated components. Accordingly, substantially pure polypeptidesinclude those derived from eukaryotic organisms but synthesized in E.coli or other prokaryotes. By “substantially pure DNA” is meant DNA thatis free of the genes which, in the naturally-occurring genome of theorganism from which the DNA of the invention is derived, flank the gene.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector; into an autonomously replicating plasmid orvirus; or into the genomic DNA of a prokaryote or eukaryote; or whichexists as a separate molecule (e.g., a cDNA or a genomic or cDNAfragment produced by PCR or restriction endonuclease digestion)independent of other sequences. It also includes a recombinant DNA whichis part of a hybrid gene encoding additional polypeptide sequence.

By “transformed cell” is meant a cell into which (or into an ancestor ofwhich) has been introduced, by means of recombinant DNA techniques, aDNA molecule encoding (as used herein) an IAP polypeptide.

By “transgene” is meant any piece of DNA which is inserted by artificeinto a cell, and becomes part of the genome of the organism whichdevelops from that cell. Such a transgene may include a gene which ispartly or entirely heterologous (i.e., foreign) to the transgenicorganism, or may represent a gene homologous to an endogenous gene ofthe organism.

By “transgenic” is meant any cell which includes a DNA sequence which isinserted by artifice into a cell and becomes part of the genome of theorganism which develops from that cell. As used herein, the transgenicorganisms are generally transgenic mammalian (e.g., rodents such as ratsor mice) and the DNA (transgene) is inserted by artifice into thenuclear genome.

By “transformation” is meant any method for introducing foreignmolecules into a cell. Lipofection, calcium phosphate precipitation,retroviral delivery, electroporation, and biolistic transformation arejust a few of the teachings which may be used. For example, biolistictransformation is a method for introducing foreign molecules into a cellusing velocity driven microprojectiles such as tungsten or goldparticles. Such velocity-driven methods originate from pressure burstswhich include, but are not limited to, helium-driven, air-driven, andgunpowder-driven techniques. Biolistic transformation may be applied tothe transformation or transfection of a wide variety of cell types andintact tissues including, without limitation, intracellular organelles(e.g., and mitochondria and chloroplasts), bacteria, yeast, fungi,algae, animal tissue, and cultured cells.

By “positioned for expression” is meant that the DNA molecule ispositioned adjacent to a DNA sequence which directs transcription andtranslation of the sequence (i.e., facilitates the production of, e.g.,an IAP polypeptide, a recombinant protein or a RNA molecule).

By “reporter gene” is meant a gene whose expression may be assayed; suchgenes include, without limitation, glucuronidase (GUS), luciferase,chloramphenicol transacetylase (CAT), and β-galactosidase.

By “promoter” is meant minimal sequence sufficient to directtranscription. Also included in the invention are those promoterelements which are sufficient to render promoter-dependent geneexpression controllable for cell type-specific, tissue-specific orinducible by external signals or agents; such elements may be located inthe 5′ or 3′ regions of the native gene.

By “operably linked” is meant that a gene and one or more regulatorysequences are connected in such a way as to permit gene expression whenthe appropriate molecules (e.g., transcriptional activator proteins arebound to the regulatory sequences).

By “conserved region” is meant any stretch of six or more contiguousamino acids exhibiting at least 30%, preferably 50%, and most preferably70% amino acid sequence identity between two or more of the IAP familymembers, (e.g., between human HIAP-1, HIAP-2, and XIAP).

Examples of preferred conserved regions are shown (as boxed ordesignated sequences) in FIGS. 5-7 and Tables 1 and 2, and include,without limitation, BIR domains and ring zinc finger domains.

By “detectably-labelled” is meant any means for marking and identifyingthe presence of a molecule, e.g., an oligonucleotide probe or primer, agene or fragment thereof, or a cDNA molecule. Methods fordetectably-labelling a molecule are well known in the art and include,without limitation, radioactive labelling (e.g., with an isotope such as³²P or ³⁵S) and nonradioactive labelling (e.g., chemiluminescentlabelling, e.g., fluorescein labelling).

By “antisense,” as used herein in reference to nucleic acids, is meant anucleic acid sequence, regardless of length, that is complementary tothe coding strand of a gene.

By “purified antibody” is meant antibody which is at least 60%, byweight, free from proteins and naturally occurring organic moleculeswith which it is naturally associated. Preferably, the preparation is atleast 75%, more preferably 90%, and most preferably at least 99%, byweight, antibody, e.g., an IAP specific antibody. A purified antibodymay be obtained, for example, by affinity chromatography usingrecombinantly-produced protein or conserved motif peptides and standardtechniques.

By “specifically binds” is meant an antibody that recognizes and binds aprotein but that does not substantially recognize and bind othermolecules in a sample, e.g., a biological sample, that naturallyincludes protein.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G show the human xiap cDNA sequence (SEQ ID NO:3) and the XIAPpolypeptide sequence (SEQ ID NO:4).

FIGS. 2A-2G show the human hiap-1 cDNA sequence (SEQ ID NO:5) and theHIAP-1 polypeptide sequence (SEQ ID NO:6).

FIGS. 3A-3G show the human hiap-2 cDNA sequence (SEQ ID NO:7) and theHIAP-2 polypeptide sequence (SEQ ID NO:8). The sequence absent in thehiap-2-Δ variant is boxed.

FIGS. 4A-4F show the murine xiap cDNA sequence (SEQ ID NO:9) and encodedmurine XIAP polypeptide sequence (SEQ ID NO:10).

FIGS. 5A-5F show FIG. 5 is the murine hiap-1 cDNA sequence (SEQ IDNO:39) and the encoded murine HIAP-1 polypeptide sequence (SEQ IDNO:40).

FIGS. 6A-6F show the murine hiap-2 cDNA sequence (SEQ ID NO:41) and theencoded murine HIAP-2 polypeptide (SEQ ID NO:42).

FIG. 7 is a representation of the alignment of the BIR domains of IAPproteins (SEQ ID NOs 11 and 14-31).

FIGS. 8A-8E show FIG. 8 is a representation of the alignment of humanIAP polypeptides with diap, cp-iap, and the IAP consensus sequence (SEQID NOs:4, 6, 8, 10, 12, and 13).

FIG. 9 is a representation of the alignment of the ring zinc fingerdomains of IAP proteins (SEQ ID NOs:32-38).

FIGS. 10A-10C are photographs of Northern blots illustrating humanhiap-1 and hiap-2 mRNA expression in human tissues.

FIGS. 11A-11C are photographs of Northern blots illustrating humanhiap-2 mRNA expression in human tissues.

FIGS. 12A-12C are photographs of Northern blots illustrating human xiapmRNA expression in human tissues.

FIGS. 13A and 13B are photographs of agarose gels illustrating apoptoticDNA ladders and RT-PCR products using hiap-1 and hiap-2 specific probesin HIV-infected T cells.

FIGS. 14A-14C are graphs depicting suppression of apoptosis by XIAP,HIAP-1, HIAP-2, bcl-2, smn, and 6-myc.

FIGS. 15A-15B are bar graphs depicting the percentage of viable CHOcells following transient transfection with the cDNA constructs shownand subsequent serum withdrawal.

FIGS. 16A-16B are bar graphs depicting the percentage of viable CHOcells following transient transfection with the cDNA constructs shownand subsequent exposure to menadione (FIG. 16A=10 μM menadione; FIG.16B=20 μM menadione).

FIG. 17 is a photograph of an agarose gel containing cDNA fragments thatwere amplified, with hiap-1-specific primers, from RNA obtained fromRaji, Ramos, EB-3, and Jiyoye cells, and from normal placenta.

FIG. 18 is a photograph of a Western blot containing protein extractedfrom Jurkat and astrocytoma cells stained with an anti-XIAP antibody.The position and size of a series of marker proteins is indicated.

FIG. 19 is a photograph of a Western blot containing protein extractedfrom Jurkat cells following treatment as described in Example XII. Theblot was stained with a rabbit polyclonal anti-XIAP antibody. Lane 1,negative control; lane 2, anti-Fas antibody; lane 3, anti-Fas antibodyand cycloheximide; lane 4, TNF-α; lane 5, TNF-α and cycloheximide.

FIG. 20 is a photograph of a Western blot containing protein extractedfrom HeLa cells following exposure to anti-Fas antibodies. The blot wasstained with a rabbit polyclonal anti-XIAP antibody. Lane 1, negativecontrol; lane 2, cycloheximide; lane 3, anti-Fas antibody; lane 4,anti-Fas antibody and cycloheximide; lane 5, TNF-α; lane 6, TNF-α andcycloheximide.

FIGS. 21A-21B are photographs of Western blots stained with rabbitpolyclonal anti-XIAP antibody. Protein was extracted from HeLa cells(FIG. 21A) and Jurkat cells (FIG. 21B) immediately, 1, 2, 3, 5, 10, and22 hours after exposure to anti-Fas antibody.

FIGS. 22A and 22B are photographs of Western blots stained with ananti-CPP32 antibody (FIG. 22A) or a rabbit polyclonal anti-XIAP antibody(FIG. 22B). Protein was extracted from Jurkat cells immediately, 3hours, or 7 hours after exposure to an anti-Fas antibody. In addition tototal protein, cytoplasmic and nuclear extracts are shown.

FIG. 23 is a photograph of a polyacrylamide gel followingelectrophoresis of the products of an in vitro XIAP cleavage assay.

DETAILED DESCRIPTION I. IAP Genes and Polypeptides

A new class of mammalian proteins that modulate apoptosis (IAPS) and thegenes that encode these proteins have been discovered. The IAP proteinsare characterized by the presence of a ring zinc finger domain (RZF;FIG. 9) and at least one BIR domain, as defined by the boxed consensussequences shown in FIGS. 7 and 8A-E, and by the sequence domains listedin Tables 1 and 2. As examples of novel IAP genes and proteins, the cDNAsequences and amino acid sequences for human IAPs (HIAP-1, HIAP-2, andXIAP) and a new murine inhibitor of apoptosis, XIAP, are provided.Additional members of the mammalian IAP family (including homologs fromother species and mutant sequences) may be isolated using standardcloning techniques and the conserved amino acid sequences, primers, andprobes provided herein and known in the art. Furthermore, IAPs includethose proteins lacking the ring zinc finger, as further described below.

TABLE 1 NUCLEOTIDE POSITION OF CONSERVED DOMAINS* Ring Zinc BIR-1 BIR-2BIR-3 Finger h-xiap 109-312 520-723  826-1023 1348-1485 m-xiap 202-405613-816  916-1113 1438-1575 h-hiap-1 273-476 693-893  951-1154 1824-1961m-hiap-1 251-453 670-870  928-1131 1795-1932 h-hiap-2 373-576 787-9871042-1245 1915-2052 m-hiap-2 215-418 608-808  863-1066 1763-1876*Positions indicated correspond to those shown in FIGS. 1-4.

TABLE 2 AMINO ACID POSITION OF CONSERVED DOMAINS* Ring Zinc BIR-1 BIR-2BIR-3 Finger h-XAIP 26-93 163-230 265-330 439-484 m-XIAP 26-93 163-230264-329 438-483 h-HIAP1 29-96 169-235 255-322 546-591 m-HIAP1 29-96169-235 255-322 544-589 h-HIAP2 46-113 184-250 269-336 560-605 m-HIAP225-92 156-222 241-308 541-578 *Positions indicated correspond to thoseshown in FIGS. 1-4.

Recognition of the mammalian IAP family has provided an emergent patternof protein structure. Recognition of this pattern allows proteins havinga known, homologous sequence but unknown function to be classified asputative inhibitors of apoptosis. A drosophila gene, now termed diap,was classified in this way (for sequence information see GenbankAccession Number M96581 and FIGS. 6A-F). The conservation of theseproteins across species indicates that the apoptosis signalling pathwayhas been conserved throughout evolution.

The IAP proteins may be used to inhibit the apoptosis that occurs aspart of numerous disease processes or disorders. For example, IAPpolypeptides or nucleic acid encoding IAP polypeptides may beadministered for the treatment or prevention of apoptosis that occurs asa part of AIDS, neurodegenerative diseases, ischemic injury,toxin-induced liver disease and myelodysplastic syndromes. Nucleic acidencoding the IAP polypeptide may also be provided to inhibit apoptosis.

II. Cloning of IAP Genes

A. xiap

The search for human genes involved in apoptosis resulted in theidentification of an X-linked sequence tag site (STS) in the GenBankdatabase, which demonstrated strong homology with the conserved RZFdomain of CPIAP and OpIAP, the two baculovirus genes known to inhibitapoptosis (Clem et al., Mol. Cell Biol. 14:5212-5222, 1994; Birnbaum etal., J. Virol. 68:2521-8, 1994). Screening a human fetal brain ZapIIcDNA library (Stratagene, La Jolla, Calif.) with this STS resulted inthe identification and cloning of xiap (for X-linked Inhibitor ofApoptosis Protein gene). The human gene has a 1.5 kb coding sequencethat includes three BIR domains is (Crook et al., J. Virol. 67:2168-74,1993; Clem et al., Science 254:1388-90, 1991; Birnbaum et al., J.Virol., 68:2521-8, 1994) and a zinc finger. Northern blot. analysis withxiap revealed message greater than 7 kb, which is expressed in varioustissues, particularly liver and kidney (FIG. 12). The large size of thetranscript reflects large 5′ and 3′ untranslated regions.

B.Human hiap-1 and hiap-2

The hiap-1 and hiap-2 genes were cloned by screening a human liverlibrary (Stratagene Inc., LaJolla, Calif.) with a probe including theentire xiap coding region at low stringency (the final wash wasperformed at 40° C. with 2×SSC, 10% SDS; FIGS. 2A-G and 3A-G). Thehiap-1 and hiap-2 genes were also detected independently using a probederived from an expressed sequence tag (EST; GenBank Accession No.T96284), which includes a portion of a BIR domain. The EST sequence wasoriginally isolated by the polymerase chain reaction; a cDNA library wasused as a template and amplified with EST-specific primers. The DNAampliderived probe was then used to screen the human liver cDNA libraryfor full-length hiap coding sequences. A third DNA was subsequentlydetected that includes the hiap-2 sequence but that appears to lack oneexon, presumably due to alternative mRNA splicing (see boxed region in3A-3G). The expression of hiap-1 and hiap-2 in human tissues as assayedby Northern blot analysis is shown in FIGS. 8A-8E and 9.

C. M-xiap

Fourteen cDNA and two genomic clones were identified by screening amouse embryo λgt11 cDNA library (Clontech, Palo Alto, Calif.) and amouse FIX II genomic library with a xiap cDNA probe, respectively. AcDNA contig spanning 8.0 kb was constructed using 12 overlapping mouseclones. Sequence analysis revealed a coding sequence of approximately1.5 kb. The mouse gene, m-xiap, encodes a polypeptide with strikinghomology to human Xiap at and around the initiation methionine, the stopcodon, the three BIR domains, and the RZF domain. As with the humangene, the mouse homologue contains large 5′ and 3′ UTRS, which couldproduce a transcript as large as 7-8 kb.

Analysis of the sequence and restriction map of m-xiap further delineatethe structure and genomic organization of m-xiap. Southern blot analysisand inverse PCR techniques (Groden et al., Cell 66:589-600, 1991) can beemployed to map exons and define exon-intron boundaries.

Antisera can be raised against a m-xiap fusion protein that was obtainedfrom, for example, E. coli using a bacterial expression system. Theresulting antisera can be used along with Northern blot analysis toanalyze the spatial and temporal expression of m-xiap in the mouse.

D. m-hiap-1 and m-hiap-2

The murine homologs of hiap-1 and hiap-2 were cloned and sequenced inthe same general manner as m-xiap using the human hiap-1 and hiap-2sequences as probes. cloning of m-hiap-1 and m-hiap-2 furtherdemonstrate that homologs from different species may be isolated usingthe techniques provided herein and those generally known to artisansskilled in molecular biology.

III. Identification of Additional IAP Genes

Standard techniques, such as the polymerase chain reaction (PCR) and DNAhybridization, may be used to clone additional human IAP genes and theirhomologues in other species. Southern blots of human genomic DNAhybridized at low stringency with probes specific for xiap, hiap-1 andhiap-2 reveal bands that correspond to other known human IAP sequencesas well as additional bands that do not correspond to known IAPsequences. Thus, additional IAP sequences may be readily identifiedusing low stringency hybridization. Examples of murine and human xiap,hiap-1, and hiap-2 specific primers, which may be used to cloneadditional genes by RT-PCR, are shown in Table 4.

IV. Characterization of IAP Activity and Intracellular LocalizationStudies

The ability of putative IAPs to modulate apoptosis can be defined in invitro systems in which alterations of apoptosis can be detected.Mammalian expression constructs carrying IAP cDNAs, which are eitherfull-length or truncated, can be introduced into cell lines such as CHO,NIH 3T3, HL60, Rat-1, or Jurkat cells. In addition, SF21 insect cellsmay be used, in which case the IAP gene is preferentially expressedusing an insect heat shock promotor. Following transfection, apoptosiscan be induced by standard methods, which include serum withdrawal, orapplication of staurosporine, menadione (which induces apoptosis viafree radial formation), or anti-Fas antibodies. As a control, cells arecultured under the same conditions as those induced to undergoapoptosis, but either not transfected, or transfected with a vector thatlacks an IAP insert. The ability of each IAP construct to inhibitapoptosis upon expression can be quantified by calculating the survivalindex of the cells, i.e., the ratio of surviving transfected cells tosurviving control cells. These experiments can confirm the presence ofapoptosis inhibiting activity and, as discussed below, can also be usedto determine the functional region(s) of an IAP. These assays may alsobe performed in combination with the application of additional compoundsin order to identify compounds that modulate apoptosis via IAPexpression.

Cell Survival Following Transfection with Full-length IAP Constructs andInduction of Apoptosis

Specific examples of the results obtained by performing various apoptois suppression assays are shown in FIGS. 14A to 14D. For example, CHOcell survival following transfection with one of six constructs andsubsequent serum withdrawal is shown in FIG. 14A. The cells weretransfected using Lipofectace™ with 2 μg of one of the followingrecombinant plasmids: pCDNA36myc-xiap (xiap), pCDNA3-6myc-hiap-1(hiap-1), pCDNA3-6myc-hiap-2 (hiap-2), pCDNA3-bcl-2 (bcl-2),pCDNA3-HA-smn (smn), and pCDNA3-6myc (6-myc.). Oligonucleotide primerswere synthesized to allow PCR amplification and cloning of the xiap,hiap-1, and hiap-2 ORFs in pCDNA3 (Invitrogen). Each construct wasmodified to incorporate a synthetic myc tag encoding six repeats of thepeptide sequence MEQKLISEEDL (SEQ ID NO: 43), thus allowing detection ofmyc-IAP fusion proteins via monoclonal anti-myc antiserum (Egan et al.,Nature 363:45, 1993). Triplicate samples of cell lines in 24-well disheswere washed 5 times with serum free media and maintained in serum freeconditions during the course of the experiment. Cells that excludedtrypan blue, and that were therefore viable, were counted with ahemocytometer immediately, 24 hours, 48 hours, and 72 hours after serumwithdrawal. Survival was calculated as a percentage of the initialnumber of viable cells. In this experiment, as well as those presentedin FIGS. 14B and 14D, the percentage of viable cells shown representsthe average of three separate experiments performed in triplicate, ±standard deviation.

The survival of CHO cells following transfection (with each one of thesix constructs described above) and exposure to menadione is shown inFIG. 14B. The cells were plated in 24-well dishes, allowed to growovernight, and then exposed to 20 μM menadione for 1.5 hours (SigmaChemical Co., St. Louis, Mo.). Triplicate samples were harvested at thetime of exposure to menadione and 24 hours afterward, and survival wasassessed by trypan blue exclusion.

The survival of Rat-1 cells following transfection (with each one of thesix constructs described above) and exposure to staurosporine is shownin FIG. 14C. Rat-1 cells were transfected and then selected in mediumcontaining 800 μg/ml G418 for two weeks. The cell line was assessed forresistance to staurosporine-induced apoptosis (1 μM) for 5 hours. Viablecells were counted 24 hours after exposure to staurosporine by trypanblue exclusion. The percentage of viable cells shown represents theaverage of two experiments, ± average deviation.

The Rat-1 cell line was also used to test the resistance of these cellsto menadione (FIG. 14D) following transfection with each of the sixconstructs described above. The cells were exposed to 10 μM menadionefor 1.5 hours, and the number of viable cells was counted 18 hourslater.

B. Comparison of Cell Survival Following Transfection with Full-lengthvs. Partial IAP Constructs

In order to investigate the mechanism whereby human IAPs, includingXIAP, HIAP-1, and HIAP-2, afford protection against cell death,expression vectors were constructed that contained either: (1)full-length IAP cDNA (as described above), (2) a portion of an IAP genethat encodes the BIR domains, but not the RZF, or (3) a portion of anIAP gene that encodes the RZF, but not the BIR domains. Human and murinexiap or m-xiap cDNAs were tested by transient or stable expression inHeLa, Jurkat, and CHO cell lines. Following transfection, apoptosis wasinduced by serum withdrawal, application of menadione, or application ofan anti-Fas antibody. Cell death was then assessed, as described above,by trypan blue exclusion. As a control for transfection efficiency, thecells were co-transfected with a β-gal expression construct. Typically,approximately 20% of the cells were successfully transfected.

When CHO cells were transiently transfected, constructs containingfull-length xiap or m-xiap cDNAs conferred modest protection againstcell death (FIG. 15A). In contrast, the survival of CHO cellstransfected with constructs encoding only the BIR domains (i.e., lackingthe RZF domain; see FIG. 15A) was markedly enhanced 72 hours after serumdeprivation. Furthermore, a large percentage of cells expressing the BIRdomains were still viable after 96 hours, at which time no viable cellsremained in the control, i.e. non-transfected, cell cultures (see “CHO”in FIG. 15A), and less than 5% of the cells transfected with the vectoronly, i.e., lacking a cDNA insert, remained viable (see “pcDNA3” in FIG.15A). Deletion of any of the BIR domains results in the complete loss ofapoptotic suppression, which is reflected by a decrease in thepercentage of surviving CHO cells to control levels within 72 hours ofserum withdrawal (FIG. 15B; see “xiapΔ1” (which encodes amino acids89-497 of XIAP (SEQ ID NO.:4)), “xiapΔ2” (which encodes amino acids246-497 of XIAP (SEQ ID NO.:4)), and “xiapΔ3” (which encodes amino acids342-497 of XIAP (SEQ ID NO.:4)) at 72 hours).

Stable pools of transfected CHO cells, which were maintained for severalmonths under G418 selection, were induced to undergo apoptosis byexposure to 10 μM menadione for 2 hours. Among the CHO cells tested werethose that were stably transfected with: (1) full-length m-xiap cDNA(miap), (2) full-length xiap cDNA (xiap), (3) full-length bcl-2 cDNA(Bcl-2), (4) cDNA encoding the three BIR domains (but not the RZF) ofm-xiap (BIR), and (5) cDNA encoding the RZF (but not BIR domains) ofm-xiap (RZF). Cells that were non-transfected (CHO) or transfected withthe vector only (pcDNA3), served as controls for this experiment.Following exposure to 10 μM menadione, the transfected cells were washedwith phosphate buffered saline (PBS) and cultured for an additional 24hours in menadione-free medium. Cell death was assessed, as describedabove, by trypan blue exclusion. Less than 10% of the non-transfected orvector-only transfected cells remained viable at the end of the 24 hoursurvival period. Cells expressing the RZF did not fare significantlybetter. However, expression of full-length m-xiap, xiap, or bcl-2, andexpression of the BIR domains, enhanced cell survival (FIG. 16A). Whenthe concentration of menadione was increased from 10 μM to 20 μM (withall other conditions of the experiment being the same as when 10 μMmenadione was applied), the percentage of viable CHO cells thatexpressed the BIR domain cDNA construct was higher than the percentageof viable cells that expressed either full-length m-xiap or bcl-2 (FIG.16B).

C. Analysis of the Subcellular Location of Expressed RZF and BIR Domains

The assays of cell death described above indicate that the RZF may actas a negative regulator of the anti-apoptotic function of IAPs. One wayin which the RZF, and possibly other IAP domains, may exert theirregulatory influence is by altering the expression of genes, whoseproducts function in the apoptotic pathway.

In order to determine whether the subcellular locations of expressed RZFand BIR domains are consistent with roles as nuclear regulatory factors,COS cells were transiently transfected with the following fourconstructs, and the expressed polypeptide was localized byimmunofluorescent microscopy: (1) pcDNA3-6myc-xiap, which encodes all497 amino acids of SEQ ID NO:4, (2) pcDNA3-6myc-m-xiap, which encodesall 497 amino acids of mouse xiap (SEQ ID NO:10), (3)pcDNA3-6myc-mxiap-BIR, which encodes amino acids 1 to 341 of m-xiap (SEQID NO:10), and (4) pcDNA3-6myc-mxiap-RZF, which encodes amino acids342-497 of m-xiap (SEQ ID NO:10). The cells were grown on multi-welltissue culture slides for 12 hours, and then fixed and permeabilizedwith methanol.

The constructs used (here and in the cell death assays) were tagged witha human Myc epitope tag at the N-terminus. Therefore, a monoclonalanti-Myc antibody and a secondary goat anti-mouse antibody, which wasconjugated to FITC, could be used to localize the expressed products intransiently transfected COS cells. Full-length XIAP and MIAP werelocated in the cytoplasm, with accentuated expression in theperi-nuclear zone. The same pattern of localization was observed whenthe cells expressed a construct encoding the RZF domain (but not the BIRdomains). However, cells expressing the BIR domains (without the RZF)exhibited, primarily, nuclear staining. The protein expressed by the BIRdomain construct appeared to be in various stages of transfer to thenucleus.

These observations are consistent with the fact that, as describedbelow, XIAP is cleaved within T cells that are treated with anti-Fasantibodies (which are potent inducers of apoptosis), and its N-terminaldomain is translocated to the nucleus.

D. Examples of Additional Apoptosis Assays

Specific examples of apoptosis assays are also provided in the followingreferences. Assays for apoptosis in lymphocytes are disclosed by: Li etal., “Induction of apoptosis in uninfected lymphocytes by HIV-1 Tatprotein”;, Science 268:429-431, 1995; Gibellini et al., “Tat-expressingJurkat cells show an increased resistance to different apoptoticstimuli, including acute human immunodeficiency virus-type 1′ (HIV-1)infection”, Br. J. Haematol. 89:24-33, 1995; Martin et al., “HIV-1infection of human CD4⁺ T cells in vitro. Differential induction ofapoptosis in these cells.” J. Immunol. 152:330-42, 1994; Terai et al.,“Apoptosis as a mechanism of cell death in cultured T lymphoblastsacutely infected with-HIV-1”, J. Clin Invest. 87:1710-5, 1991; Dhein etal., “Autocrine T-cell suicide mediated by APO-l/(Fas/CD95)11, Nature373:438-441, 1995; Katsikis et al., “Fas antigen stimulation inducesmarked apoptosis of T lymphocytes in human immunodeficiencyvirus-infected individuals”, J. Exp. Med. 1815:2029-2036, 1995;Westendorp et al., Sensitization of T cells to CD95-mediated apoptosisby HIV-1 Tat and gp120”, Nature 375:497, 1995; DeRossi et al., Virology198:234-44, 1994.

Assays for apoptosis in fibroblasts are disclosed by: Vossbeck et al.,“Direct transforming activity of TGF-beta on rat fibroblasts”, Int. J.Cancer 61:92-97, 1995; Goruppi et al., “Dissection of c-myc domainsinvolved in S phase induction of NIH3T3 fibroblasts”, Oncogene9:1537-44, 1994; Fernandez et al., “Differential sensitivity of normaland Ha-ras transformed C3H mouse embryo fibroblasts to tumor necrosisfactor: induction of bcl-2, c-myc, and manganese superoxide dismutase inresistant cells”, Oncogene 9:2009-17, 1994; Harrington et al.,“c-Myc-induced apoptosis in fibroblasts is inhibited by specificcytokines”, EMBO J., 13:3286-3295, 1994; Itoh et al., “A novel proteindomain required for apoptosis. Mutational analysis of human Fasantigen”, J. Biol. Chem. 268:10932-7, 1993.

Assays for apoptosis in neuronal cells are disclosed by: Melino et al.,“Tissue transglutaminase and apoptosis: sense and antisense transfectionstudies with human neuroblastoma cells”, Mol. Cell Biol. 14:6584-6596,1994; Rosenbaumet al., “Evidence for hypoxia-induced, programmed celldeath of cultured neurons”, Ann. Neurol. 36:864-870, 1994; Sato et al.,“Neuronal differentiation of PC12 cells as a result of prevention ofcell death by bcl-2”, J. Neurobiol 25:1227-1234, 1994; Ferrari et al.,“N-acetylcysteine D- and L-stereoisomers prevents apoptotic death ofneuronal cells”, J. Neurosci. 1516:2857-2866, 1995; Talley et al.,“Tumor necrosis factor alpha-induced apoptosis in human neuronal cells:protection by the antioxidant N-acetylcysteine and the genes bcl-2 andcrma”, Mol. Cell Biol. 1585:2359-2366, 1995; Talley et al., “TumorNecrosis Factor Alpha-Induced Apoptosis in Human Neuronal Cells:Protection by the Antioxidant NAcetylcysteine and the Genes bcl-2 andcrma”, Mol. Cell. Biol. 15:2359-2366, 1995; Walkinshaw et al.,“Induction of apoptosis in catecholaminergic PC12 cells by L-DOPA.Implications for the treatment of Parkinson's disease.”, J. Clin.Invest. 95:2458-2464, 1995.

Assays for apoptosis in insect cells are disclosed by: Clem et al.,“Prevention of apoptosis by a baculovirus gene during infection ofinsect cells”, Science 254:1388-90, 1991; Crook et al., “Anapoptosis-inhibiting baculovirus gene with a zinc finger-like motif”, J.Virol. 67:2168-74, 1993; Rabizadeh et al., “Expression of thebaculovirus p35 gene inhibits mammalian neural cell death”, J.Neurochem. 61:2318-21, 1993; Birnbaum et al., “An apoptosis inhibitinggene from a nuclear polyhedrosis virus encoding a polypeptide withCys/His sequence motifs”, J. Virol. 68:2521-8, 1994; Clem et al.,“Control of programmed cell death by the baculovirus genes p35 and IAP”,Mol. Cell. Biol. 14:5212-5222, 1994.

V. Construction of a Transgenic Animal

Characterization of IAP genes provides information that is necessary foran IAP knockout animal model to be developed by homologousrecombination. Preferably, the model is a mammalian animal, mostpreferably a mouse. Similarily, an animal model of IAP overproductionmay be generated by integrating one or more IAP sequences into thegenome, according to standard transgenic techniques.

A replacement-type targeting vector, which would be used to create aknockout model, can be constructed using an isogenic genomic clone, forexample, from a mouse strain such as 129/Sv (Stratagene Inc., LaJolla,Calif.). The targeting vector will be introduced into a suitably-derivedline of embryonic stem (ES) cells by electroporation to generate ES celllines that carry a profoundly truncated form of an IAP. To generatechimeric founder mice, the targeted cell lines will be injected into amouse blastula stage embryo. Heterozygous offspring will be interbred tohomozygosity. Knockout mice would provide the means, in vivo, to screenfor therapeutic compounds that modulate apoptosis via an IAP-dependentpathway.

VI. IAP Protein Expression

IAP genes may be expressed in both prokaryotic and eukaryotic celltypes. If an IAP modulates apoptosis by exacerbating it, it may bedesirable to express that protein under control of an induciblepromotor.

In general, IAPs according to the invention may be produced bytransforming a suitable host cell with all or part of an IAP-encodingcDNA fragment that has been placed into a suitable expression vector.

Those skilled in the art of molecular biology will understand that awide variety of expression systems may be used to produce therecombinant protein. The precise host cell used is not critical to theinvention. The IAP protein may be produced in a prokaryotic host (e.g.,E. coli) or in a eukaryotic host (e.g., S. cerevisiae, insect cells suchas Sf2l cells, or mammalian cells such as COS-1, NIH 3T3, or HeLacells). These cells are publically available, for example, from theAmerican Type Culture Collection, Rockville, Md.; see also Ausubel etal., Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, N.Y., 1994). The method of transduction and the choice ofexpression vehicle will depend on the host system selected.Transformation and transfection methods are described, e.g., in Ausubelet al. (supra), and expression vehicles may be chosen from thoseprovided, e.g. in Cloning Vectors: A Laboratory Manual (P. H. Pouwels etal., 1985, Supp. 1987).

A preferred expression system is the baculovirus system using, forexample, the vector pBacPAK9, which is available from Clontech (PaloAlto, Calif.). If desired, this system may be used in conjunction withother protein expression techniques, for example, the myc tag approachdescribed by Evan et al. (Mol. Cell Biol. 5:3610-3616, 1985).

Alternatively, an IAP may be produced by a stably-transfected mammaliancell line. A number of vectors suitable for stable transfection ofmammalian cells are available to the public, e.g., see Pouwels et al.(supra), as are methods for constructing such cell lines (see e.g.,Ausubel et al. (supra). In one example, cDNA encoding an IAP is clonedinto an expression vector that includes the dihydrofolate reductase(DHFR) gene. Integration of the plasmid and, therefore, integration ofthe IAP-encoding gene into the host cell chromosome is selected for byinclusion of 0.01-300 μM methotrexate in the cell culture medium (asdescribed, Ausubel et al., supra). This dominant selection can beaccomplished in most cell types. Recombinant protein expression can beincreased by DHFR-mediated amplification of the transfected gene.

Methods for selecting cell lines bearing gene amplifications aredescribed in Ausubel et al. (supra). These methods generally involveextended culture in medium containing gradually increasing levels ofmethotrexate. The most commonly used DHFR-containing expression vectorsare PCVSEII-DHFR and pAdD26SV(A) (described in Ausubel et al., supra).The host cells described above or, preferably, a DHFR-deficient CHO cellline (e.g., CHO DHFR cells, ATCC Accession No. CRL 9096) are among thosemost preferred for DHFR selection of a stably-transfected cell line orDHFR-mediated gene amplification.

Once the recombinant protein is expressed, it is isolated by, forexample, affinity chromatography. In one example, an anti-IAP antibody,which may be produced by the methods described herein, can be attachedto a column and used to isolate the IAP protein. Lysis and fractionationof IAP-harboring cells prior to affinity chromatography may be performedby standard methods (see e.g., Ausubel et al., supra). Once isolated,the recombinant protein can, if desired, be purified further by e.g., byhigh performance liquid chromatography (HPLC; e.g., see Fisher,Laboratory Techniques In Biochemistry And Molecular Biology, Work andBurdon, Eds., Elsevier, 1980).

Polypeptides of the invention, particularly short IAP fragments, canalso be produced by chemical synthesis (e.g., by the methods describedin Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co.,Rockford, Ill.). These general techniques of polypeptide expression andpurification can also be used to produce and isolate useful IAPfragments or analogs, as described herein.

VII. Anti-IAP Antibodies

In order to generate IAP-specific antibodies, an IAP coding sequence(i.e., amino acids 180-276) can be expressed as a C-terminal fusion withglutathione S-transferase (GST; Smith et al., Gene 67:31-40, 1988). Thefusion protein can be purified on glutathione-Sepharose beads, elutedwith glutathione, and cleaved with thrombin (at the engineered cleavagesite), and purified to the degree required to successfully immunizerabbits. Primary immunizations can be carried out with Freund's completeadjuvant and subsequent immunizations performed with Freund's incompleteadjuvant. Antibody titres are monitored by Western blot andimmunoprecipitation analyses using the thrombin-cleaved IAP fragment ofthe GST-IAP fusion protein. Immune sera are affinity purified usingCNBr-Sepharose-coupled IAP protein. Antiserum specificity is determinedusing a panel of unrelated GST proteins (including GSTp53, Rb, HPV-16E6, and E6-AP) and GST-trypsin (which was generated by PCR using knownsequences).

As an alternate or adjunct immunogen to GST fusion proteins, peptidescorresponding to relatively unique hydrophilic regions of IAP may begenerated and coupled to keyhole limpet hemocyanin (KLH) through anintroduced C-terminal lysine. Antiserum to each of these peptides issimilarly affinity purified on peptides conjugated to BSA, andspecificity is tested by ELISA and Western blotting using peptideconjugates, and by Western blotting and immunoprecipitation using IAPexpressed as a GST fusion protein.

Alternatively, monoclonal antibodies may be prepared using the IAPproteins described above and standard hybridoma technology (see, e.g.,Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol.6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling etal., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, New York,N.Y., 1981; Ausubel et al., supra). Once produced, monoclonal antibodiesare also tested for specific IAP recognition by Western blot orimmunoprecipitation analysis (by the methods described in Ausubel etal., supra).

Antibodies that specifically recognize IAPs or fragments of IAPs, suchas those described herein containing one or more BIR domains (but not aring zinc finger domain), or that contain a ring zinc finger domain (butnot a BIR domain) are considered useful in the invention. They may, forexample, be used in an immunoassay to monitor IAP expression levels orto determine the subcellular location of an IAP or IAP fragment producedby a mammal. Antibodies that inhibit the 26 kDa IAP cleavage productdescribed herein (which contains at least one BIR domain) may beespecially useful in inducing apoptosis in cells undergoing undesirableproliferation.

Preferably, antibodies of the invention are produced using IAP sequencethat does not reside within highly conserved regions, and that appearslikely to be antigenic, as analyzed by criteria such as those providedby the Peptide structure program (Genetics computer Group SequenceAnalysis Package, Program Manual for the GCG Package, Version 7, 1991)using the algorithm of Jameson and Wolf (CABIOS 4:181, 1988).Specifically, these regions, which are found between BIR1 and BIR2 ofall IAPs, are: from amino acid 99 to amino acid 170 of hiap-1, fromamino acid 123 to amino acid 184 of hiap-2, and from amino acid 116 toamino acid 133 of either xiap or m-xiap. These fragments can begenerated by standard techniques, e.g. by the PCR, and cloned into thepGEX expression vector (Ausubel et al., supra). Fusion proteins areexpressed in E. coli and purified using a glutathione agarose affinitymatrix as described in Ausubel et al. (supra). In order to minimize thepotential for obtaining antisera that is non-specific, or exhibitslow-affinity binding to IAP, two or three fusions are generated for eachprotein, and each fusion is injected into at least two rabbits. Antiseraare raised by injections in series, preferably including at least threebooster injections.

VIII. Identification of Molecules that Modulate IAP Protein Expression

Isolation of IAP cDNAs also facilitates the identification of moleculesthat increase or decrease IAP expression. In one approach, candidatemolecules are added, in varying concentration, to the culture medium ofcells expressing IAP mRNA. IAP expression is then measured, for example,by Northern blot analysis (Ausubel et al., supra) using an IAP cDNA, orcDNA fragment, as a hybridization probe (see also Table 5). The level ofIAP expression in the presence of the candidate molecule is compared tothe level of IAP expression in the absence of the candidate molecule,all other factors (e.g. cell type and culture conditions) being equal.

The effect of candidate molecules on IAP-mediated apoptosis may,instead, be measured at the level of translation by using the generalapproach described above with standard protein detection techniques,such as Western blotting or immunoprecipitation with an IAP-specificantibody (for example, the IAP antibody described herein).

Compounds that modulate the level of IAP may be purified, orsubstantially purified, or may be one component of a mixture ofcompounds such as an extract or supernatant obtained from cells (Ausubelet al., supra). In an assay of a mixture of compounds, IAP expression istested against progressively smaller subsets of the compound pool (e.g.,produced by standard purification techniques such as HPLC or FPLC) untila single compound or minimal number of effective compounds isdemonstrated to modulate IAP expression.

Compounds may also be screened for their ability to modulate IAPapoptosis inhibiting activity. In this approach, the degree of apoptosisin the presence of a candidate compound is compared to the degree ofapoptosis in its absence, under equivalent conditions. Again, the screenmay begin with a pool of candidate compounds, from which one or moreuseful modulator compounds are isolated in a step-wise fashion.Apoptosis activity may be measured by any standard assay, for example,those described herein.

Another method for detecting compounds that modulate the activity ofIAPs is to screen for compounds that interact physically with a givenIAP polypeptide. These compounds may be detected by adapting interactiontrap expression systems known in the art. These systems detect proteininteractions using a transcriptional activation assay and are generallydescribed by Gyuris et al. (Cell 75:791-803, 1993) and Field et al.,Nature 340:245-246, 1989), and are commercially available from Clontech(Palo Alto, Calif.). In addition, PCT Publication WO 95/28497 describesan interaction trap assay in which proteins involved in apoptosis, byvirtue of their interaction with Bcl-2, are detected. A similar methodmay be used to identify proteins and other compounds that interact withIAPs.

Compounds or molecules that function as modulators of IAP-mediated celldeath may include peptide and non-peptide molecules such as thosepresent in cell is extracts, mammalian serum, or growth medium in whichmammalian cells have been cultured.

A molecule that promotes an increase in IAP expression or IAP activityis considered particularly useful in the invention; such a molecule maybe used, for example, as a therapeutic to increase cellular levels ofIAP and thereby exploit the ability of IAP polypeptides to inhibitapoptosis.

A molecule that decreases IAP activity (e.g., by decreasing IAP geneexpression or polypeptide activity) may be used to decrease cellularproliferation. This would be advantageous in the treatment of neoplasms(see Table 3, below), or other cell proliferative diseases.

TABLE 3 NORTHERN BLOT IAP RNA LEVELS IN CANCER CELLS* xiap hiap1 hiap2Promyelocytic Leukemia HL-60 + + + Hela S-3 + + + Chronic MyelogenousLeukemia K-562 +++ + +++ Lymphoblastic Leukemia MOLT-4 +++ + + Burkitt'sLymphoma Raji + +(x10) + Colorectal Adenocarcinoma SW-480 +++ +++ +++Lung Carcinoma A-549 + + + Melanoma G-361 +++ + + *Levels are indicatedby a (+) and are the approximate increase in RNA levels relative toNorthern blots of RNA from non-cancerous control cell lines. A singleplus indicates an estimated increase of at least 1-fold

Molecules that are found, by the methods described above, to effectivelymodulate IAP gene expression or polypeptide activity may be testedfurther in animal models. If they continue to function successfully inan in vivo setting, they may be used as therapeutics to either inhibitor enhance apoptosis, as appropriate.

IX. IAP Therapy

The level of IAP gene expression correlates with the level of apoptosis.Thus, IAP genes also find use in anti-apoptosis gene therapy. Inparticular, a functional IAP gene may be used to sustain neuronal cellsthat undergo apoptosis in the course of a neurodegenerative disease,lymphocytes (i.e., T cells and B cells), or cells that have been injuredby ischemia.

Retroviral vectors, adenoviral vectors, adeno-associated viral vectors,or other viral vectors with the appropriate tropism for cells likely tobe involved in apoptosis (for example, epithelial cells) may be used asa gene transfer delivery system for a therapeutic IAP gene construct.Numerous vectors useful for this purpose are generally known (Miller,Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989;Eglitis and Anderson, BioTechniques 6:608-614, 1988; Tolstoshev andAnderson, current opinion in Biotechnology 1:55-61, 1990; Sharp, TheLancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research andMolecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984;Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechniques7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; andJohnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularlywell developed and have been used in clinical settings (Rosenberg etal., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No.5,399,346). Non-viral approaches may also be employed for theintroduction of therapeutic DNA into cells otherwise predicted toundergo apoptosis. For example, IAP may be introduced into a neuron or aT cell by lipofection (Felgner et al., Proc. Natl. Acad. Sci. USA84:7413, 1987; Ono et al., Neurosci. Lett. 117:259, 1990; Brigham etal., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Meth. Enz.101:512, 1983), asialorosonucoid-polylysine conjugation. (Wu et al., J.Biol. Chem. 263:14621, 1988; Wu et al., J. Biol. Chem. 264:16985, 1989);or, less preferably, microinjection under surgical conditions (Wolff etal., Science 247:1465, 1990).

For any of the methods of application described above, the therapeuticIAP DNA construct is preferably applied to the site of the predictedapoptosis event (for example, by injection). However, it may also beapplied to tissue in the vicinity of the predicted apoptosis event or toa blood vessel supplying the cells predicted to undergo apoptosis.

In the constructs described, IAP cDNA expression can be directed fromany suitable promoter (e.g., the human cytomegalovirus (CMV), simianvirus 40 (SV40), or metallothionein promoters), and regulated by anyappropriate mammalian regulatory element. For example, if desired,enhancers known to preferentially direct gene expression in neuralcells, T cells, or B cells may be used to direct IAP expression. Theenhancers used could include, without limitation, those that arecharacterized as tissue- or cell-specific in their expression.Alternatively, if an IAP genomic clone is used as a therapeuticconstruct (for example, following its isolation by hybridization withthe IAP cDNA described above), regulation may be mediated by the cognateregulatory sequences or, if desired, by regulatory sequences derivedfrom a heterologous source, including any of the promoters or regulatoryelements described above.

Less preferably, IAP gene therapy is accomplished by directadministration of the IAP mRNA or antisense IAP mRNA to a cell that isexpected to undergo apoptosis. The mRNA may be produced and isolated byany standard technique, but is most readily produced by in vitrotranscription using an IAP cDNA under the control of a high efficiencypromoter (e.g., the T7 promoter). Administration of IAP mRNA tomalignant cells can be carried out by any of the methods for directnucleic acid administration described above.

Ideally, the production of IAP protein by any gene therapy approach willresult in cellular levels of IAP that are at least equivalent to thenormal, cellular level of IAP in an unaffected cell. Treatment by anyIAP-mediated gene therapy approach may be combined with more traditionaltherapies.

Another therapeutic approach within the invention involvesadministration of recombinant IAP protein, either directly to the siteof a predicted apoptosis event (for example, by injection) orsystemically (for example, by any conventional recombinant proteinadministration technique). The dosage of IAP depends on a number offactors, including the size and health of the individual patient, but,generally, between 0.1 mg and 100 mg inclusive are administered per dayto an adult in any pharmaceutically-acceptable formulation.

X. Administration of IAP Polypeptides, IAP Genes, or Modulators of IAPSynthesis or Function

An IAP protein, gene, or modulator may be administered within apharmaceutically-acceptable diluent, carrier, or excipient, in unitdosage form. Conventional pharmaceutical practice may be employed toprovide suitable formulations or compositions to administer IAP topatients suffering from a disease that is caused by excessive apoptosis.Administration may begin before the patient is symptomatic. Anyappropriate route of administration may be employed, for example,administration may be parenteral, intravenous, intraarterial,subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic,intraventricular, intracapsular, intraspinal, intracisternal,intraperitoneal, intranasal, aerosol, or oral administration.Therapeutic formulations may be in the form of liquid solutions orsuspensions; for oral administration, formulations may be in the form oftablets or capsules; and for intranasal formulations, in the form ofpowders, nasal drops, or aerosols.

Methods well known in the art for making formulations are found, forexample, in “Remington's Pharmaceutical Sciences.” Formulations forparenteral administration may, for example, contain excipients, sterilewater, or saline, polyalkylene glycols such as polyethylene glycol, oilsof vegetable origin, or hydrogenated napthalenes. Biocompatible,biodegradable lactide polymer, lactide/glycolide copolymer, orpolyoxyethylene-polyoxypropylene copolymers may be used to control therelease of the compounds. Other potentially useful parenteral deliverysystems for IAP modulatory compounds include ethylene-vinyl acetatecopolymer particles, osmotic pumps, implantable infusion systems, andliposomes. Formulations for inhalation may contain excipients, forexample, lactose, or may be aqueous solutions containing, for example,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may beoily solutions for administration in the form of nasal drops, or as agel.

If desired, treatment with an IAP protein, gene, or modulatory compoundmay be combined with more traditional therapies for the disease such assurgery, steroid therapy, or chemotherapy for autoimmune disease;antiviral therapy for AIDS; and tissue plasminogen activator (TPA) forischemic injury.

XI. Detection of Conditions Involving Altered Apoptosis

IAP polypeptides and nucleic acid sequences find diagnostic use in thedetection or monitoring of conditions involving aberrant levels ofapoptosis. For example, decrease expression of IAP may be correlatedwith enhanced apoptosis in humans (see XII, below). Accordingly, adecrease or increase in the level of IAP production may provide anindication of a deleterious condition. Levels of IAP expression may beassayed by any standard technique. For example, IAP expression in abiological sample (e.g., a biopsy) may be monitored by standard Northernblot analysis or may be aided by PCR (see, e.g., Ausubel et al., supra;PCR Technology: Principles and Applications for DNA Amplification, H. A.Ehrlich, Ed. Stockton Press, NY; Yap et al. Nucl. Acids. Res. 19:4294,1991).

Alternatively, a biological sample obtained from patient may be analyzedfor one or more mutations in he IAP sequences using a mismatch detectionapproach. Generally, these techniques involve PCR amplification ofnucleic acid from the patient sample, followed by identification of themutation (i.e., mismatch) by either. altered hybridization, aberrantelectrophoretic gel migration, binding or cleavage mediated by mismatchbinding proteins, or direct nucleic acid sequencing. Any of thesetechniques may be used to facilitate mutant IAP detection, and each iswell known in the art; examples of particular techniques are described,without limitation, in Orita et al., Proc. Natl. Acad. Sci. USA86:2766-2770, 1989; Sheffield et al., Proc. Natl. Acad. Sci. USA86:232-236, 1989).

In yet another approach, immunoassays are used to detect or monitor IAPprotein in a biological sample. IAPspecific polyclonal or monoclonalantibodies (produced as described above) may be used in any standardimmunoassay format (e.g., ELISA, Western blot, or RIA) to measure IAPpolypeptide levels. These levels would be compared to wild-type IAPlevels, with a decrease in IAP production indicating a conditioninvolving increased apoptosis. Examples of immunoassays are described,e.g., in Ausubel et al., supra. Immunohistochemical techniques may alsobe utilized for IAP detection. For example, a tissue sample may beobtained from a patient, sectioned, and stained for the presence of IAPusing an anti-IAP antibody and any standard detection system (e.g., onewhich includes a secondary antibody conjugated to horseradishperoxidase). General guidance regarding such techniques can be found in,e.g., Bancroft and Stevens (Theory and Practice of HistologicalTechniques, Churchill Livingstone, 1982) and Ausubel et al. (supra).

In one preferred example, a combined diagnostic method may be employedthat begins with an evaluation of IAP protein production (for example,by immunological techniques or the protein truncation test (Hogerrorstet al., Nature Genetics 10:208-212, 1995) and also includes a nucleicacid-based detection technique designed to identify more subtle IAPmutations (for example, point mutations). As described above, a numberof mismatch detection assays are available to those skilled in the art,and any preferred technique may be used. Mutations in IAP may bedetected that either result in loss of IAP expression or loss of IAPbiological activity. In a variation of this combined diagnostic method,IAP biological activity is measured as protease activity using anyappropriate protease assay system (for example, those described above).

Mismatch detection assays also provide an opportunity to diagnose anIAP-mediated predisposition to diseases caused by inappropriateapoptosis. For example, a patient heterozygous for an IAP mutation mayshow no clinical symptoms and yet possess a higher than normalprobability of developing one or more types of neurodegenerative,myelodysplastic or ischemic diseases. Given this diagnosis, a patientmay take precautions to minimize their exposure to adverse environmentalfactors (for example, UV exposure or chemical mutagens) and to carefullymonitor their medical condition (for example, through frequent physicalexaminations). This type of IAP diagnostic approach may also be used todetect IAP mutations in prenatal screens. The IAP diagnostic assaysdescribed above may be carried out using any biological sample (forexample, any biopsy sample or bodily fluid or tissue) in which IAP isnormally expressed. Identification of a mutant IAP gene may also beassayed using these sources for test samples.

Alternatively, a IAP mutation, particularly as part of a diagnosis forpredisposition to IAP-associated degenerative disease, may be testedusing a DNA sample from any cell, for example, by mismatch detectiontechniques. Preferably, the DNA sample is subjected to PCR amplificationprior to analysis.

In order to demonstrate the utility of IAP gene sequences as diagnosticsand prognostics for cancer, a Human Cancer Cell Line Multiple TissueNorthern Blot (Clontech, Palo Alto, Calif.; #7757-1) was probed. ThisNorthern blot contained approximately 2 μg of poly A⁺ RNA per lane fromeight different human cell lines: (1) promyelocytic leukemia HL-60, (2)HeLa cell S3, (3) chronic myelogenous-leukemia K-562, (4) lymphoblasticleukemia MOLT-4, (5) Burkitt's lymphoma Raji, (6) colorectaladenocarcinoma SW480, (7) lung carcinoma A549, and (8) melanoma G361. Asa control, a Human Multiple Tissue Northern Blot (Clontech, Palo Alto,Calif.; #17759-1) was probed. This Northern blot contained approximately2 μg of poly A⁺ RNA from eight different human tissues: (1) spleen, (2)thymus, (3) prostate, (4) testis, (5) ovary, (6) small intestine, (7)colon, and (8) peripheral blood leukocytes.

The Northern blots were hybridized sequentially with: (1) a 1.6 kb probeto the xiap coding region, (2) a 375 bp hiap-2 specific probecorresponding to the 3′ untranslated region, (3) a 1.3 kb probe to thecoding region of hiap-1, which cross-reacts with hiap-2, (4) a 1.0 kbprobe derived from the coding region of bcl-2, and (5) a probe toβ-actin, which was provided by the manufacturer. Hybridization wascarried out at 50° C. overnight, according to the manufacturer'ssuggestion.

The blot was washed twice with 2×SSC, 0.1% SDS at room temperature for15 minutes and then with 2×SSC, 0.1% SDS at 50° C.

All cancer lines tested showed increased IAP expression relative tosamples from non-cancerous control tissues (Table 3). Expression of xiapwas particularly high in HeLa (S-3), chronic myelogenous leukemia(K-562), colorectal adenocarcinoma (SW-480), and melanoma (G-361) lines.Expression of hiap-1 was extremely high in Burkitt's lymphoma, and wasalso elevated in colorectal adenocarcinoma. Expression of hiap-2 wasparticularly high in chronic myelogenous leukemia (K-562) and colorectaladenocarcinoma (SW-480). Expression of Bcl-2 was upregulated only inHL-60 leukemia cells.

These observations suggest that upregulation of the anti-apoptotic IAPgenes may be a widespread phenomenon, perhaps occurring much morefrequently than upregulation of Bcl-2. Furthermore, upregulation may benecessary for the establishment or maintenance of the transformed stateof cancerous cells.

In order to pursue the observation described above, i.e., that hiap-1 isoverexpressed in the Raji Burkitt's lymphoma cell line, RT-PCR analysiswas performed in multiple Burkitt's lymphoma cell lines. Total RNA wasextracted from cells of the Raji, Ramos, EB-3, and Jiyoye cell lines,and as a positive control, from normal placental tissue. The RNA wasreverse transcribed, and amplified by PCR with the following set ofoligonucleotide primers: 5′-AGTGCGGGTTTTTATTATGTG-3′ (SEQ ID NO: 44) and5′-AGATGACCACAAGGAATAAACACTA-3′ (SEQ ID NO: 45), which selectivelyamplify a hiap-1 cDNA fragment. RT-PCR was conducted using a PerkinElmer 480 Thermocycler to carry out 35 cycles of the following program:94° C. for 1 minute, 50° C. for 1.5 minutes, and 72° C. for a minute.The PCR reaction product was electrophoresed on an agarose gel andstained with ethidium bromide. Amplified cDNA fragments of theappropriate size were clearly visible in all lanes containing Burkitt'slymphoma samples, but absent in the lanes containing the normalplacental tissue sample, and absent in lanes containing negative controlsamples, where template DNA was omitted from the reaction (FIG. 17).

XII. Accumulation of a 26 kDa Cleavage Protein in Astrocytoma Cells

A. Identification of a 26 kDa Cleavage Protein

A total protein extract was prepared from Jurkat and astrocytoma cellsby sonicating them (X3 for 15 seconds at 4° C.) in 50 mM Tris-HCl (pH8.0), 150 mM NaCl, 1 mM PMSF, 1 μg/ml aprotinin, and 5 mM benzamidine.Following sonication, the samples were centrifuged (14,000 RPM in amicrofuge) for five minutes. Twenty μg of protein was loaded per well ona 10% SDS-polyacrylamide gel, electrophoresed, and electroblotted bystandard methods to PVDF membranes. Western blot analysis, performed asdescribed previously, revealed that the astrocytoma cell line(CCF-STTG1) abundantly expressed an anti-xiap reactive band ofapproximately 26 kDa, despite the lack of an apoptotic trigger event(FIG. 18). In fact, this cell line has been previously characterized asbeing particularly resistant to standard apoptotic triggers.

A 26 kDa xiap-reactive band was also observed under the followingexperimental conditions. Jurkat cells (a transformed human T cell line)were induced to undergo apoptosis by exposure to an anti-Fas antibody (1μg/ml). Identical cultures of Jurkat cells were exposed either to: (1)anti-Fas antibody and cycloheximide (20 μg/ml), (2) tumor necrosisfactor alpha (TNF-α, at 1,000 U/ml), or (3) TNF-α and cycloheximide (20μg/ml). All cells were harvested 6 hours after treatment began. Inaddition, as a negative control, anti-Fas antibody was added to anextract after the cells were harvested. The cells were harvested in SDSsample buffer, electrophoresed on a 12.5% SDS polyacrylamide gel, andelectroblotted onto PVDF membranes using standard methods. The membraneswere immunostained with a rabbit polyclonal anti-XIAP antibody at 1:1000for 1 hour at room temperature. Following four 15 minute washes, a goatanti-rabbit antibody conjugated to horse-radish peroxidase was appliedat room temperature for 1 hour. Unbound secondary antibody was washedaway, and chemiluminescent detection of XIAP protein was performed. TheWestern blot revealed the presence of the full-length, 55 kDa XIAPprotein, both in untreated and treated cells. In addition, a novel,approximately 26 kDa xiap-reactive band was also observed in apoptoticcell extracts, but not in the control, untreated cell extracts (FIG.19).

Cleavage of XIAP occurs in a variety of cell types, including othercancer cell lines such as HeLa. The expression of the 26 kDa XIAPcleavage product was demonstrated in HeLa cells as follows. HeLa cellswere treated with either: (1) cyclohexamide (20 μg/ml), (2) anti-Fasantibody (1 μg/ml), (3) anti-Fas antibody (1 μg/ml) and cyclohexamide(20 μg/ml), (4) TNFα (1,000 U/ml), or (5) TNFα (1,000 U/ml) andcyclohexamide (20 μg/ml). All cells were harvested 18 hours aftertreatment began. As above, anti-Fas antibody was added to an extractafter the cells were harvested. HeLa cells were harvested, and theWestern blot was probed under the same conditions as used to visualizexiap-reactive bands from Jurkat cell samples. A 26 kDa XIAP band wasagain seen in the apoptotic cell preparations (FIG. 20). Furthermore,the degree of XIAP cleavage correlated positively with the extent ofapoptosis. Treatment of HeLa cells with cycloheximide or TNFα alonecaused only minor apoptosis, and little cleavage product was observed.If the cells were treated with the anti-Fas antibody, a greater amountof cleavage product was apparent. These data indicate that XIAP iscleaved in more than one cell type and in response to more than one typeof apoptotic trigger.

B. Time Course of Expression

The time course over which the 26 kDa cleavage product accumulates wasexamined by treating HeLa and Jurkat cells with anti-Fas antibody (1μg/ml) and harvesting them either immediately, or 1, 2, 3, 5, 10, or 22hours after treament. Protein extracts were prepared and Western blotanalysis was performed as described above. Both types of cellsaccumulated increasing quantities of the 26 kDa cleavage product overthe time course examined (FIGS. 21A and 21B).

C. Subcellular Localization of the 26 kDa XIAP Cleavage Product

In order to determine the subcellular location of the 26 kDa cleavageproduct, Jurkat cells were induced to undergo apoptosis by exposure toanti-Fas antibody (1 μg/ml) and were then harvested either immediately,3 hours, or 7 hours later. Total protein extracts were prepared, asdescribed above, from cells harvested at each time point. In order toprepare nuclear and cytoplasmic cell extracts, apoptotic Jurkat cellswere washed with isotonic Tris buffered saline (pH 7.0) and lysed byfreezing and thawing five times in cell extraction buffer (50 mM PIPES,50 mM KCl, 5 mM EGTA, 2 mM MgCl₂, λ mM DTT, and 20 μM cytochalasin B).Nuclei were pelleted by centrifugation and resuspended in isotonic Tris(pH 7.0) and frozen at −80° C. The cytoplasmic fraction of the extractwas processed further by centrifugation at 60,000 RPM in a TA 100.3rotor for 30 minutes. Supernatants were removed and frozen at −80° C.Samples of both nuclear and cytoplasmic fractions were loaded on a 12.5%SDS-polyacrylamide gel, and electroblotted onto PVDF membranes. Westernblot analysis was then performed using either an anti-CPP32 antibody(Transduction Laboratories Lexington, Ky.; FIG. 22A) or the rabbitanti-XIAP antibody described above (FIG. 22B).

The anti-CPP32 antibody, which recognizes the CPP32 protease (also knownas YAMA or Apopain) partitioned almost exclusively in the cytoplasmicfraction. The 55 kDa XIAP protein localized exclusively in the cytoplasmof apoptotic cells, in agreement with the studies presented above, whereXIAP protein in normal, healthy COS cells was seen to localize, byimmunofluoresence microscopy, to the cytoplasm. In contrast, the 26 kDacleavage product localized exclusively to the nuclear fraction ofapoptotic Jurkat cells. Taken together, these observations suggest thatthe anti-apoptotic component of XIAP could be the 26 kDa cleavageproduct, which exerts its influence within the nucleus.

D. In Vitro Cleavage of XIAP Protein and Characterization of theCleavage Product

For this series of experiments, XIAP protein was labeled with ³⁵S usingthe plasmid pcDNA3-6myc-XIAP, T7 RNA polymerase, and a coupledtranscription/translation kit (Promega) according to the manufacturer'sinstructions. Radioactively labeled XIAP protein was separated fromunincorporated methionine by column chromatography using Sephadex G-50.In addition, extracts of apoptotic Jurkat cells were prepared followingtreatment with anti-Fas antibody (1 μg/ml) for three hours. To preparethe extracts, the cells were lysed in Triton X-100 buffer (1% TritonX-100, 25 mM Tris HCl) on ice for two hours and then microcentrifugedfor 5 minutes. The soluble extract was retained (and was labeled TX100).Cells were lysed in cell extraction buffer with freeze/thawing. Thesoluble cytoplasmic fraction was set aside (and labeled CEB). Nuclearpellets from the preparation of the CEB cytoplasmic fraction weresolubilized with Triton X-100 buffer, microcentrifuged, and the solublefractions, which contains primarily nuclear DNA, was retained (andlabeled CEB-TX100). Soluble cell extract was prepared by lysing cellswith NP-40 buffer, followed by microcentrifugation for 5 minutes (andwas labeled NP-40). In vitro cleavage was performed by incubating 16 μlof each extract (CEB, TX-100, CEB-TX100, and NP-40) with 4 μl of invitro translated XIAP protein at 37° C. for 7 hours. Negative controls,containing only TX100 buffer or CEB buffer were also included. Theproteins were separated on a 10% SDS-polyacrylamide gel, which was driedand exposed to X-ray film overnight.

In vitro cleavage of XIAP was apparent in the CEB extract. The observedmolecular weight of the cleavage product was approximately 36 kDa (FIG.23). The 10 kDa shift in the size of the cleavage product indicates thatthe observed product is derived from the amino-terminus of therecombinant protein, which contains six copies of the myc epitope (10kDa). It thus appears that the cleavage product possesses at least twoof the BIR domains, and that it is localized to the nucleus.

XIII. Treatment of HIV Infected Individuals

The expression of hiap-1 and hiap-2 is decreased significantly inHIV-infected human cells. Furthermore, this decrease precedes apoptosis.Therefore, administration of HIAP-1, HIAP-2, genes encoding theseproteins, or compounds that upregulate these genes can be used toprevent T cell attrition in HIV-infected patients. The following assaymay also be used to screen for compounds that alter hiap-1 and hiap-2expression, and which also prevent apoptosis.

Cultured mature lymphocyte CD-4+T cell lines (H9, labelled “a”;CEM/CM-3, labelled “b”; 6T-CEM, labelled “c”; and Jurkat, labelled “d”in FIGS. 13A and 13B), were examined for signs of apoptosis (FIG. 13A)and hiap gene expression (FIG. 13B) after exposure to mitogens or HIVinfection. Apoptosis was demonstrated by the appearance of DNA“laddering” upon gel electrophoresis and gene expression was assessed byPCR. The results obtained from normal (non-infected, non-mitogenstimulated) cells are shown in each lane labelled “1” in FIGS. 13A and13B. The results obtained. 24 hours after PHA/PMA(phytohemagglutinin/phorbol ester) stimulation are shown in each lanelabelled “2”. The results obtained 24 hours after HIV strain IIIBinfection are shown in each lane labelled “3”. The “M” refers tostandard DNA markers (the 123 bp ladder in FIG. 13B, and the lambdaHindIII ladder in FIG. 13A (both from Gibco-BRL)). DNA ladders (Prigentet al., J. Immunol. Methods, 160:139-140, 1993), which indicateapoptosis, are evident when DNA from the samples described above areelectrophoresed on an ethidium bromide-stained agarose gel (FIG. 13A).The sensitivity and degree of apoptosis of the four T cell lines testedvaries following mitogen stimulation and HIV infection.

In order to examine hiap gene expression, total RNA was prepared fromthe cultured cells and reverse transcribed using oligo-dT priming. TheRT cDNA products were amplified by PCR using specific primers (as shownin Table 5) for the detection of hiap-2a, hiap-2b and hiap-1. The PCRwas conducted using a PerkinElmer 480 thermocycler with 35 cycles of thefollowing program: 94° C. for one minute, 55° C. for 2 minutes and 72°C. for 1.5 minutes. The RT-PCR reaction products were electrophoresed ona 1% agarose gel, which was stained with ethidium bromide. Absence ofhiap-2 transcripts is noted in all four cell lines 24 hours after HIVinfection. In three of four cell lines (all except H9), the hiap-1 geneis also dramatically down-regulated after HIV infection. PHA/PMA mitogenstimulation also appears to decrease hiap gene expression; particularlyof hiap-2 and to a lesser extent, of hiap-1. The data from theseexperiments is summarized in Table 5. The expression of β-actin wasconsistent in all cell lines tested, indicating that there is not a flawin the RT-PCR assay that could account for the decrease in hiap geneexpression.

TABLE 4 OLIGONUCLEOTIDE PRIMERS FOR THE SPECIFIC RT-PCR AMPLIFICATION OFUNIQUE IAP GENES Forward Primer Reverse Primer Size of XAP (nucleotide(nucleotide Product Gene position*) position*) (bp) h-xiap p2415 (876-896) p2449 (1291-1311) 435 m-xiap p2566  (458-478) p2490 (994-1013) 555 h-hiap1 p2465  (827-847) p2464 (1008-1038) 211 m-hiap1p2687  (747-767) p2684 (1177-1197) 450 hiap2 p2595 (1562-1585) p2578(2339-2363) 801^(a) 618^(b) m-hiap2 p2693 (1751-1772) p2734 (2078-2100)349 *Nucleotide position as determined from FIGS. 1-4 for each IAP gene^(a)PCR product size of hiap2a ^(b)PCR product size of hiap2b

TABLE 5 APOPTOSIS AND HIAP GENE EXPRESSION IN CULTURED T-CELLS FOLLOWINGMITOGEN STIMULATION OR HIV INFECTION Cell Line condition Apoptosis hiap1hiap2 H9 not stimulated − + ± PHA/PMA stimulated +++ + ± HIV infected++ + − CEM/CM-3 not stimulated − + ± PHA/PMA stimulated ± + − HIVinfected ± − − 6T-CEM not stimulated − + + PHA/PMA stimulated ± − − HIVinfected + − − Jurkat not stimulated − + ++ PHA/PMA stimulated + + + HIVinfected ± − −

XIV. Assignment of xiap, hiap-1, and hiap-2 to Chromosomes Xq25 and11q22-23 by Fluorescence in situ Hybridization

(FISH)

Fluorescence in situ hybridization (FISH) was used to identify thechromosomal location of xiap, hiap-1 and hiap-2. The probes used werecDNAs cloned in plasmid vectors: the 2.4 kb xiap clone included 1493 bpof coding sequence, 34bp of 5′ UTR (untranslated region) and 913 bp of3′UTR; the hiap-1 cDNA was 3.1 kb long and included 1812 bp coding and1300 bp of 3′ UTR; and the hiap-2 clone consisted of 1856 bp of codingand 1200 bp of 5′ UTR. A total of 1 μg of probe DNA was labelled withbiotin by nick translation (BRL). Chromosome spreads prepared from anormal peripheral blood culture were denatured for 2 minutes at 70° C.in 50% formamide/2×SSC and subsequently hybridized with the biotinlabelled DNA probe for 18 hours at 370° C. in a solution consisting of2×SSC/70% formamide/10% dextran sulfate. After hybridization, thespreads were washed in 2×SSC/50% formamide, followed by a wash in 2×SSCat 42° C. The biotin labelled DNA was detected by fluoresceinisothiocyanate (FITC) conjugated avidin antibodies and anti-avidinantibodies (ONCOR detection kit), according to the manufacturer'sinstructions. Chromosomes were counterstained with propidium iodide andexamined with a Olympus BX60 epifluorescence microscope. For chromosomeidentification, the slides with recorded labelled metaphase spreads weredestained, dehydrated, dried, digested with trypsin for 30 seconds andstained with 4% Giemsa stain for 2 minutes. The chromosome spreads wererelocated and the images were compared.

A total of 101 metaphase spreads were examined with the xiap probe, asdescribed above. Symmetrical fluorescent signals on either one or bothhomologs of chromosome Xq25 were observed in 74% of the cells analyzed.Following staining with hiap-1 and hiap-2 probes, 56 cells were analyzedand doublet signals in the region 11q22-23 were observed in 83% of cellsexamined. The xiap gene was mapped to Xq25 while the hiap-1 and hiap-2genes were mapped at the border of 11q22 and 11q23 bands.

These experiments confirmed the location of the xiap gene on chromosomeXq25. No highly consistent chromosomal abnormalities involving band Xq25have been reported so far in any malignancies. However, deletions withinthis region are associated with a number of immune system defectsincluding X-linked lymphoproliferative disease (Wu et al., Genomics17:163-170, 1993).

Cytogenetic abnormalities of band 11q23 have been identified in morethan 50% of infant leukemias regardless of the phenotype(Martinez-Climet et al., Leukaemia 9:1299-1304, 1995). Rearrangements ofthe MLL Gene (mixed lineage leukemia or myeloid lymphoid leukemia;Ziemin Van der Poel et al., Proc. Natl. Acad. Sci. USA 88:10735-10739,1991) have been detected in 80% of cases with 11q23 translocation,however patients whose rearrangements clearly involved regions otherthan the MLL gene were also reported (Kobayashi et al., Blood82:547-551, 1993). Thus, the IAP genes may follow the Bcl-2 paradigm,and would therefore play an important role in cancer transformation.

XV. Preventive Anti-Apoptotic Therapy

In a patient diagnosed to beheterozygous for an IAP mutation or to besusceptible to IAP mutations (even if those mutations do not yet resultin alteration or loss of IAP biological activity), or a patientdiagnosed as HIV positive, any of the above therapies may beadministered before the occurrence of the disease phenotype. Forexample, the therapies may be provided to a patient who is HIV positivebut does not yet show a diminished T cell count or other overt signs ofAIDS. In particular, compounds shown to increase IAP expression or IAPbiological activity may be administered by any standard dosage and routeof administration (see above). Alternatively, gene therapy using an IAPexpression construct may be undertaken to reverse or prevent the celldefect prior to the development of the degenerative disease.

The methods of the instant invention may be used to reduce or diagnosethe disorders described herein in any mammal, for example, humans,domestic pets, or livestock. Where a non-human mammal is treated ordiagnosed, the IAP polypeptide, nucleic acid, or antibody employed ispreferably specific for that species.

Other Embodiments

In other embodiments, the invention includes any protein which issubstantially identical to a mammalian IAP polypeptides. (FIGS. 1-6; SEQID NOs:1-42); such homologs include other substantially purenaturally-occurring mammalian IAP proteins as well as allelic variants;natural mutants; induced mutants; DNA sequences which encode proteinsand also hybridize to the IAP DNA sequences of FIGS. 1-6 (SEQ IDNOS:1-42) under high stringency conditions or, less preferably, underlow stringency conditions (e.g., washing at 2×SSC at 400° C. with aprobe length of at least 40 nucleotides); and proteins specificallybound by antisera directed to a IAP polypeptide. The term also includeschimeric polypeptides that include a IAP portion.

The invention further includes analogs of any naturally-occurring IAPpolypeptide. Analogs can differ from the naturally-occurring IAP proteinby amino acid sequence differences, by post-translational modifications,or by both. Analogs of the invention will generally exhibit at least85%, more preferably 90%, and most preferably 95% or even 99% identitywith all or part of a naturally occurring IAP amino acid sequence. Thelength of sequence comparison is at least 15 amino acid residues,preferably at least 25 amino acid residues, and more preferably morethan 35 amino acid residues. Modifications include in vivo and in vitrochemical derivatization of polypeptides, e.g., acetylation,carboxylation, phosphorylation, or glycosylation; such modifications mayoccur during polypeptide synthesis or processing or following treatmentwith isolated modifying enzymes. Analogs can also differ from thenaturally-occurring IAP polypeptide by alterations in primary sequence.These include genetic variants, both natural and induced (for example,resulting from random mutagenesis by irradiation or exposure toethanemethylsulfate or by site-specific mutagenesis as described inSambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual(2d ed.), CSH Press, 1989, or Ausubel et al., supra). Also included arecyclized peptides, molecules, and analogs which contain residues otherthan L-amino acids, e.g., D-amino acids or non-naturally occurring orsynthetic amino acids, e.g., B or y amino acids. In addition tofull-length polypeptides, the invention also includes IAP polypeptidefragments. As used herein, the term “fragment,” means at least 20contiguous amino acids, preferably at least 30 contiguous amino acids,more preferably at least 50 contiguous amino acids, and most preferablyat least 60 to 80 or more contiguous amino acids. Fragments of IAPpolypeptides can be generated by methods known to those skilled in theart or may result from normal protein processing (e.g., removal of aminoacids from the nascent polypeptide that are not required for biologicalactivity or removal of amino acids by alternative mRNA splicing oralternative protein processing events).

Preferable fragments or analogs according to the invention are thosewhich facilitate specific detection of a IAP nucleic acid or amino acidsequence in a sample to be diagnosed. Particularly useful IAP fragmentsfor this purpose include, without limitation, the amino acid fragmentsshown in Table 2.

45 1 46 PRT Artificial Sequence Synthetic based on Homo sapiens, Musmusculus, Drosophila melanogaster, Cydia pomonella, and Orgyiapseudotsugata 1 Glu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Lys XaaCys Met 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Pro Cys Gly HisXaa Xaa Xaa 20 25 30 Cys Xaa Xaa Cys Ala Xaa Xaa Xaa Xaa Xaa Cys Pro XaaCys 35 40 45 2 68 PRT Artificial Sequence VARIANT (1)...(66) Xaa atpositions 1, 2, 3, 6, 9, 10, 14, 15, 18, 19, 20, 21, 24, 30, 32, 33, 35,37, 40, 42, 43, 44, 45, 46, 47, 49, 50, 51, 53, 54, 55, 56, 57, 59, 60,61, 62, 64 and 66 may be any amino acid. 2 Xaa Xaa Xaa Arg Leu Xaa ThrPhe Xaa Xaa Trp Pro Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa LeuAla Xaa Ala Gly Phe Tyr Tyr Xaa Gly Xaa 20 25 30 Xaa Asp Xaa Val Xaa CysPhe Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Trp 35 40 45 Xaa Xaa Xaa Asp Xaa XaaXaa Xaa Xaa His Xaa Xaa Xaa Xaa Pro Xaa 50 55 60 Cys Xaa Phe Val 65 32540 DNA Homo sapiens variation (2540)...(2540) N may be any nucleotide3 gaaaaggtgg acaagtccta ttttcaagag aagatgactt ttaacagttt tgaaggatct 60aaaacttgtg tacctgcaga catcaataag gaagaagaat ttgtagaaga gtttaataga 120ttaaaaactt ttgctaattt tccaagtggt agtcctgttt cagcatcaac actggcacga 180gcagggtttc tttatactgg tgaaggagat accgtgcggt gctttagttg tcatgcagct 240gtagatagat ggcaatatgg agactcagca gttggaagac acaggaaagt atccccaaat 300tgcagattta tcaacggctt ttatcttgaa aatagtgcca cgcagtctac aaattctggt 360atccagaatg gtcagtacaa agttgaaaac tatctgggaa gcagagatca ttttgcctta 420gacaggccat ctgagacaca tgcagactat cttttgagaa ctgggcaggt tgtagatata 480tcagacacca tatacccgag gaaccctgcc atgtattgtg aagaagctag attaaagtcc 540tttcagaact ggccagacta tgctcaccta accccaagag agttagcaag tgctggactc 600tactacacag gtattggtga ccaagtgcag tgcttttgtt gtggtggaaa actgaaaaat 660tgggaacctt gtgatcgtgc ctggtcagaa cacaggcgac actttcctaa ttgcttcttt 720gttttgggcc ggaatcttaa tattcgaagt gaatctgatg ctgtgagttc tgataggaat 780ttcccaaatt caacaaatct tccaagaaat ccatccatgg cagattatga agcacggatc 840tttacttttg ggacatggat atactcagtt aacaaggagc agcttgcaag agctggattt 900tatgctttag gtgaaggtga taaagtaaag tgctttcact gtggaggagg gctaactgat 960tggaagccca gtgaagaccc ttgggaacaa catgctaaat ggtatccagg gtgcaaatat 1020ctgttagaac agaagggaca agaatatata aacaatattc atttaactca ttcacttgag 1080gagtgtctgg taagaactac tgagaaaaca ccatcactaa ctagaagaat tgatgatacc 1140atcttccaaa atcctatggt acaagaagct atacgaatgg ggttcagttt caaggacatt 1200aagaaaataa tggaggaaaa aattcagata tctgggagca actataaatc acttgaggtt 1260ctggttgcag atctagtgaa tgctcagaaa gacagtatgc aagatgagtc aagtcagact 1320tcattacaga aagagattag tactgaagag cagctaaggc gcctgcaaga ggagaagctt 1380tgcaaaatct gtatggatag aaatattgct atcgtttttg ttccttgtgg acatctagtc 1440acttgtaaac aatgtgctga agcagttgac aagtgtccca tgtgctacac agtcattact 1500ttcaagcaaa aaatttttat gtcttaatct aactctatag taggcatgtt atgttgttct 1560tattaccctg attgaatgtg tgatgtgaac tgactttaag taatcaggat tgaattccat 1620tagcatttgc taccaagtag gaaaaaaaat gtacatggca gtgttttagt tggcaatata 1680atctttgaat ttcttgattt ttcagggtat tagctgtatt atccattttt tttactgtta 1740tttaattgaa accatagact aagaataaga agcatcatac tataactgaa cacaatgtgt 1800attcatagta tactgattta atttctaagt gtaagtgaat taatcatctg gattttttat 1860tcttttcaga taggcttaac aaatggagct ttctgtatat aaatgtggag attagagtta 1920atctccccaa tcacataatt tgttttgtgt gaaaaaggaa taaattgttc catgctggtg 1980gaaagataga gattgttttt agaggttggt tgttgtgttt taggattctg tccattttct 2040tgtaaaggga taaacacgga cgtgtgcgaa atatgtttgt aaagtgattt gccattgttg 2100aaagcgtatt taatgataga atactatcga gccaacatgt actgacatgg aaagatgtca 2160gagatatgtt aagtgtaaaa tgcaagtggc gggacactat gtatagtctg agccagatca 2220aagtatgtat gttgttaata tgcatagaac gagagatttg gaaagatata caccaaactg 2280ttaaatgtgg tttctcttcg gggagggggg gattggggga ggggccccag aggggtttta 2340gaggggcctt ttcactttcg acttttttca ttttgttctg ttcggatttt ttataagtat 2400gtagaccccg aagggtttta tgggaactaa catcagtaac ctaacccccg tgactatcct 2460gtgctcttcc tagggagctg tgttgtttcc cacccaccac ccttccctct gaacaaatgc 2520ctgagtgctg gggcactttn 2540 4 497 PRT Homo sapiens 4 Met Thr Phe Asn SerPhe Glu Gly Ser Lys Thr Cys Val Pro Ala Asp 1 5 10 15 Ile Asn Lys GluGlu Glu Phe Val Glu Glu Phe Asn Arg Leu Lys Thr 20 25 30 Phe Ala Asn PhePro Ser Gly Ser Pro Val Ser Ala Ser Thr Leu Ala 35 40 45 Arg Ala Gly PheLeu Tyr Thr Gly Glu Gly Asp Thr Val Arg Cys Phe 50 55 60 Ser Cys His AlaAla Val Asp Arg Trp Gln Tyr Gly Asp Ser Ala Val 65 70 75 80 Gly Arg HisArg Lys Val Ser Pro Asn Cys Arg Phe Ile Asn Gly Phe 85 90 95 Tyr Leu GluAsn Ser Ala Thr Gln Ser Thr Asn Ser Gly Ile Gln Asn 100 105 110 Gly GlnTyr Lys Val Glu Asn Tyr Leu Gly Ser Arg Asp His Phe Ala 115 120 125 LeuAsp Arg Pro Ser Glu Thr His Ala Asp Tyr Leu Leu Arg Thr Gly 130 135 140Gln Val Val Asp Ile Ser Asp Thr Ile Tyr Pro Arg Asn Pro Ala Met 145 150155 160 Tyr Cys Glu Glu Ala Arg Leu Lys Ser Phe Gln Asn Trp Pro Asp Tyr165 170 175 Ala His Leu Thr Pro Arg Glu Leu Ala Ser Ala Gly Leu Tyr TyrThr 180 185 190 Gly Ile Gly Asp Gln Val Gln Cys Phe Cys Cys Gly Gly LysLeu Lys 195 200 205 Asn Trp Glu Pro Cys Asp Arg Ala Trp Ser Glu His ArgArg His Phe 210 215 220 Pro Asn Cys Phe Phe Val Leu Gly Arg Asn Leu AsnIle Arg Ser Glu 225 230 235 240 Ser Asp Ala Val Ser Ser Asp Arg Asn PhePro Asn Ser Thr Asn Leu 245 250 255 Pro Arg Asn Pro Ser Met Ala Asp TyrGlu Ala Arg Ile Phe Thr Phe 260 265 270 Gly Thr Trp Ile Tyr Ser Val AsnLys Glu Gln Leu Ala Arg Ala Gly 275 280 285 Phe Tyr Ala Leu Gly Glu GlyAsp Lys Val Lys Cys Phe His Cys Gly 290 295 300 Gly Gly Leu Thr Asp TrpLys Pro Ser Glu Asp Pro Trp Glu Gln His 305 310 315 320 Ala Lys Trp TyrPro Gly Cys Lys Tyr Leu Leu Glu Gln Lys Gly Gln 325 330 335 Glu Tyr IleAsn Asn Ile His Leu Thr His Ser Leu Glu Glu Cys Leu 340 345 350 Val ArgThr Thr Glu Lys Thr Pro Ser Leu Thr Arg Arg Ile Asp Asp 355 360 365 ThrIle Phe Gln Asn Pro Met Val Gln Glu Ala Ile Arg Met Gly Phe 370 375 380Ser Phe Lys Asp Ile Lys Lys Ile Met Glu Glu Lys Ile Gln Ile Ser 385 390395 400 Gly Ser Asn Tyr Lys Ser Leu Glu Val Leu Val Ala Asp Leu Val Asn405 410 415 Ala Gln Lys Asp Ser Met Gln Asp Glu Ser Ser Gln Thr Ser LeuGln 420 425 430 Lys Glu Ile Ser Thr Glu Glu Gln Leu Arg Arg Leu Gln GluGlu Lys 435 440 445 Leu Cys Lys Ile Cys Met Asp Arg Asn Ile Ala Ile ValPhe Val Pro 450 455 460 Cys Gly His Leu Val Thr Cys Lys Gln Cys Ala GluAla Val Asp Lys 465 470 475 480 Cys Pro Met Cys Tyr Thr Val Ile Thr PheLys Gln Lys Ile Phe Met 485 490 495 Ser 5 2676 DNA Homo sapiensvariation (2470)...(2470) N may be any nucleotide 5 tccttgagatgtatcagtat aggatttagg atctccatgt tggaactcta aatgcataga 60 aatggaaataatggaaattt ttcattttgg cttttcagcc tagtattaaa actgataaaa 120 gcaaagccatgcacaaaact acctccctag agaaaggcta gtcccttttc ttccccattc 180 atttcattatgaacatagta gaaaacagca tattcttatc aaatttgatg aaaagcgcca 240 acacgtttgaactgaaatac gacttgtcat gtgaactgta ccgaatgtct acgtattcca 300 cttttcctgctggggttcct gtctcagaaa ggagtcttgc tcgtgctggt ttctattaca 360 ctggtgtgaatgacaaggtc aaatgcttct gttgtggcct gatgctggat aactggaaaa 420 gaggagacagtcctactgaa aagcataaaa agttgtatcc tagctgcaga ttcgttcaga 480 gtctaaattccgttaacaac ttggaagcta cctctcagcc tacttttcct tcttcagtaa 540 cacattccacacactcatta cttccgggta cagaaaacag tggatatttc cgtggctctt 600 attcaaactctccatcaaat cctgtaaact ccagagcaaa tcaagaattt tctgccttga 660 tgagaagttcctacccctgt ccaatgaata acgaaaatgc cagattactt acttttcaga 720 catggccattgacttttctg tcgccaacag atctggcacg agcaggcttt tactacatag 780 gacctggagacagagtggct tgctttgcct gtggtggaaa attgagcaat tgggaaccga 840 aggataatgctatgtcagaa cacctgagac attttcccaa atgcccattt atagaaaatc 900 agcttcaagacacttcaaga tacacagttt ctaatctgag catgcagaca catgcagccc 960 gctttaaaacattctttaac tggccctcta gtgttctagt taatcctgag cagcttgcaa 1020 gtgcgggtttttattatgtg ggtaacagtg atgatgtcaa atgcttttgc tgtgatggtg 1080 gactcaggtgttgggaatct ggagatgatc catgggttca acatgccaag tggtttccaa 1140 ggtgtgagtacttgataaga attaaaggac aggagttcat ccgtcaagtt caagccagtt 1200 accctcatctacttgaacag ctgctatcca catcagacag cccaggagat gaaaatgcag 1260 agtcatcaattatccatttg gaacctggag aagaccattc agaagatgca atcatgatga 1320 atactcctgtgattaatgct gccgtggaaa tgggctttag tagaagcctg gtaaaacaga 1380 cagttcagagaaaaatccta gcaactggag agaattatag actagtcaat gatcttgtgt 1440 tagacttactcaatgcagaa gatgaaataa gggaagagga gagagaaaga gcaactgagg 1500 aaaaagaatcaaatgattta ttattaatcc ggaagaatag aatggcactt tttcaacatt 1560 tgacttgtgtaattccaatc ctggatagtc tactaactgc cggaattatt aatgaacaag 1620 aacatgatgttattaaacag aagacacaga cgtctttaca agcaagagaa ctgattgata 1680 cgattttagtaaaaggaaat attgcagcca ctgtattcag aaactctctg caagaagctg 1740 aagctgtgttatatgagcat ttatttgtgc aacaggacat aaaatatatt cccacagaag 1800 atgtttcagatctaccagtg gaagaacaat tgcggagact accagaagaa agaacatgta 1860 aagtgtgtatggacaaagaa gtgtccatag tgtttattcc ttgtggtcat ctagtagtat 1920 gcaaagattgtgctccttct ttaagaaagt gtcctatttg taggagtaca atcaagggta 1980 cagttcgtacatttctttca tgaagaagaa ccaaaacatc gtctaaactt tagaattaat 2040 ttattaaatgtattataact ttaactttta tcctaatttg gtttccttaa aatttttatt 2100 tatttacaactcaaaaaaca ttgttttgtg taacatattt atatatgtat ctaaaccata 2160 tgaacatatattttttagaa actaagagaa tgataggctt ttgttcttat gaacgaaaaa 2220 gaggtagcactacaaacaca atattcaatc caaatttcag cattattgaa attgtaagtg 2280 aagtaaaacttaagatattt gagttaacct ttaagaattt taaatatttt ggcattgtac 2340 taataccgggaacatgaagc caggtgtggt ggtatgtacc tgtagtccca ggctgaggca 2400 agagaattacttgagcccag gagtttgaat ccatcctggg cagcatactg agaccctgcc 2460 tttaaaaacnaacagnacca aanccaaaca ccagggacac atttctctgt cttttttgat 2520 cagtgtcctatacatcgaag gtgtgcatat atgttgaatc acattttagg gacatggtgt 2580 ttttataaagaattctgtga gnaaaaattt aataaagcaa ccaaattact cttaaaaaaa 2640 aaaaaaaaaaaaaaaactcg aggggcccgt accaat 2676 6 604 PRT Homo sapiens 6 Met Asn IleVal Glu Asn Ser Ile Phe Leu Ser Asn Leu Met Lys Ser 1 5 10 15 Ala AsnThr Phe Glu Leu Lys Tyr Asp Leu Ser Cys Glu Leu Tyr Arg 20 25 30 Met SerThr Tyr Ser Thr Phe Pro Ala Gly Val Pro Val Ser Glu Arg 35 40 45 Ser LeuAla Arg Ala Gly Phe Tyr Tyr Thr Gly Val Asn Asp Lys Val 50 55 60 Lys CysPhe Cys Cys Gly Leu Met Leu Asp Asn Trp Lys Arg Gly Asp 65 70 75 80 SerPro Thr Glu Lys His Lys Lys Leu Tyr Pro Ser Cys Arg Phe Val 85 90 95 GlnSer Leu Asn Ser Val Asn Asn Leu Glu Ala Thr Ser Gln Pro Thr 100 105 110Phe Pro Ser Ser Val Thr His Ser Thr His Ser Leu Leu Pro Gly Thr 115 120125 Glu Asn Ser Gly Tyr Phe Arg Gly Ser Tyr Ser Asn Ser Pro Ser Asn 130135 140 Pro Val Asn Ser Arg Ala Asn Gln Glu Phe Ser Ala Leu Met Arg Ser145 150 155 160 Ser Tyr Pro Cys Pro Met Asn Asn Glu Asn Ala Arg Leu LeuThr Phe 165 170 175 Gln Thr Trp Pro Leu Thr Phe Leu Ser Pro Thr Asp LeuAla Arg Ala 180 185 190 Gly Phe Tyr Tyr Ile Gly Pro Gly Asp Arg Val AlaCys Phe Ala Cys 195 200 205 Gly Gly Lys Leu Ser Asn Trp Glu Pro Lys AspAsn Ala Met Ser Glu 210 215 220 His Leu Arg His Phe Pro Lys Cys Pro PheIle Glu Asn Gln Leu Gln 225 230 235 240 Asp Thr Ser Arg Tyr Thr Val SerAsn Leu Ser Met Gln Thr His Ala 245 250 255 Ala Arg Phe Lys Thr Phe PheAsn Trp Pro Ser Ser Val Leu Val Asn 260 265 270 Pro Glu Gln Leu Ala SerAla Gly Phe Tyr Tyr Val Gly Asn Ser Asp 275 280 285 Asp Val Lys Cys PheCys Cys Asp Gly Gly Leu Arg Cys Trp Glu Ser 290 295 300 Gly Asp Asp ProTrp Val Gln His Ala Lys Trp Phe Pro Arg Cys Glu 305 310 315 320 Tyr LeuIle Arg Ile Lys Gly Gln Glu Phe Ile Arg Gln Val Gln Ala 325 330 335 SerTyr Pro His Leu Leu Glu Gln Leu Leu Ser Thr Ser Asp Ser Pro 340 345 350Gly Asp Glu Asn Ala Glu Ser Ser Ile Ile His Leu Glu Pro Gly Glu 355 360365 Asp His Ser Glu Asp Ala Ile Met Met Asn Thr Pro Val Ile Asn Ala 370375 380 Ala Val Glu Met Gly Phe Ser Arg Ser Leu Val Lys Gln Thr Val Gln385 390 395 400 Arg Lys Ile Leu Ala Thr Gly Glu Asn Tyr Arg Leu Val AsnAsp Leu 405 410 415 Val Leu Asp Leu Leu Asn Ala Glu Asp Glu Ile Arg GluGlu Glu Arg 420 425 430 Glu Arg Ala Thr Glu Glu Lys Glu Ser Asn Asp LeuLeu Leu Ile Arg 435 440 445 Lys Asn Arg Met Ala Leu Phe Gln His Leu ThrCys Val Ile Pro Ile 450 455 460 Leu Asp Ser Leu Leu Thr Ala Gly Ile IleAsn Glu Gln Glu His Asp 465 470 475 480 Val Ile Lys Gln Lys Thr Gln ThrSer Leu Gln Ala Arg Glu Leu Ile 485 490 495 Asp Thr Ile Leu Val Lys GlyAsn Ile Ala Ala Thr Val Phe Arg Asn 500 505 510 Ser Leu Gln Glu Ala GluAla Val Leu Tyr Glu His Leu Phe Val Gln 515 520 525 Gln Asp Ile Lys TyrIle Pro Thr Glu Asp Val Ser Asp Leu Pro Val 530 535 540 Glu Glu Gln LeuArg Arg Leu Pro Glu Glu Arg Thr Cys Lys Val Cys 545 550 555 560 Met AspLys Glu Val Ser Ile Val Phe Ile Pro Cys Gly His Leu Val 565 570 575 ValCys Lys Asp Cys Ala Pro Ser Leu Arg Lys Cys Pro Ile Cys Arg 580 585 590Ser Thr Ile Lys Gly Thr Val Arg Thr Phe Leu Ser 595 600 7 2580 DNA Homosapiens variation (2412)...(2412) N may be any nucleotide 7 ttaggttacctgaaagagtt actacaaccc caaagagttg tgttctaagt agtatcttgg 60 taattcagagagatactcat cctacctgaa tataaactga gataaatcca gtaaagaaag 120 tgtagtaaattctacataag agtctatcat tgatttcttt ttgtggtgga aatcttagtt 180 catgtgaagaaatttcatgt gaatgtttta gctatcaaac agtactgtca cctactcatg 240 cacaaaactgcctcccaaag acttttccca ggtccctcgt atcaaaacat taagagtata 300 atggaagatagcacgatctt gtcagattgg acaaacagca acaaacaaaa aatgaagtat 360 gacttttcctgtgaactcta cagaatgtct acatattcaa ctttccccgc cggggtgcct 420 gtctcagaaaggagtcttgc tcgtgctggt ttttattata ctggtgtgaa tgacaaggtc 480 aaatgcttctgttgtggcct gatgctggat aactggaaac taggagacag tcctattcaa 540 aagcataaacagctatatcc tagctgtagc tttattcaga atctggtttc agctagtctg 600 ggatccacctctaagaatac gtctccaatg agaaacagtt ttgcacattc attatctccc 660 accttggaacatagtagctt gttcagtggt tcttactcca gccttcctcc aaaccctctt 720 aattctagagcagttgaaga catctcttca tcgaggacta acccctacag ttatgcaatg 780 agtactgaagaagccagatt tcttacctac catatgtggc cattaacttt tttgtcacca 840 tcagaattggcaagagctgg tttttattat ataggacctg gagatagggt agcctgcttt 900 gcctgtggtgggaagctcag taactgggaa ccaaaggatg atgctatgtc agaacaccgg 960 aggcattttcccaactgtcc atttttggaa aattctctag aaactctgag gtttagcatt 1020 tcaaatctgagcatgcagac acatgcagct cgaatgagaa catttatgta ctggccatct 1080 agtgttccagttcagcctga gcagcttgca agtgctggtt tttattatgt gggtcgcaat 1140 gatgatgtcaaatgctttgg ttgtgatggt ggcttgaggt gttgggaatc tggagatgat 1200 ccatgggtagaacatgccaa gtggtttcca aggtgtgagt tcttgatacg aatgaaaggc 1260 caagagtttgttgatgagat tcaaggtaga tatcctcatc ttcttgaaca gctgttgtca 1320 acttcagataccactggaga agaaaatgct gacccaccaa ttattcattt tggacctgga 1380 gaaagttcttcagaagatgc tgtcatgatg aatacacctg tggttaaatc tgccttggaa 1440 atgggctttaatagagacct ggtgaaacaa acagttctaa gtaaaatcct gacaactgga 1500 gagaactataaaacagttaa tgatattgtg tcagcacttc ttaatgctga agatgaaaaa 1560 agagaagaggagaaggaaaa acaagctgaa gaaatggcat cagatgattt gtcattaatt 1620 cggaagaacagaatggctct ctttcaacaa ttgacatgtg tgcttcctat cctggataat 1680 cttttaaaggccaatgtaat taataaacag gaacatgata ttattaaaca aaaaacacag 1740 atacctttacaagcgagaga actgattgat accatttggg ttaaaggaaa tgctgcggcc 1800 aacatcttcaaaaactgtct aaaagaaatt gactctacat tgtataagaa cttatttgtg 1860 gataagaatatgaagtatat tccaacagaa gatgtttcag gtctgtcact ggaagaacaa 1920 ttgaggaggttgcaagaaga acgaacttgt aaagtgtgta tggacaaaga agtttctgtt 1980 gtatttattccttgtggtca tctggtagta tgccaggaat gtgccccttc tctaagaaaa 2040 tgccctatttgcaggggtat aatcaagggt actgttcgta catttctctc ttaaagaaaa 2100 atagtctatattttaacctg cataaaaagg tctttaaaat attgttgaac acttgaagcc 2160 atctaaagtaaaaagggaat tatgagtttt tcaattagta acattcatgt tctagtctgc 2220 tttggtactaataatcttgt ttctgaaaag atggtatcat atatttaatc ttaatctgtt 2280 tatttacaagggaagattta tgtttggtga actatattag tatgtatgtg tacctaaggg 2340 agtagcgtcnctgcttgtta tgcatcattt caggagttac tggatttgtt gttctttcag 2400 aaagctttgaanactaaatt atagtgtaga aaagaactgg aaaccaggaa ctctggagtt 2460 catcagagttatggtgccga attgtctttg gtgcttttca cttgtgtttt aaaataagga 2520 tttttctcttatttctcccc ctagtttgtg agaaacatct caataaagtg ctttaaaaag 2580 8 618 PRTHomo sapiens 8 Met His Lys Thr Ala Ser Gln Arg Leu Phe Pro Gly Pro SerTyr Gln 1 5 10 15 Asn Ile Lys Ser Ile Met Glu Asp Ser Thr Ile Leu SerAsp Trp Thr 20 25 30 Asn Ser Asn Lys Gln Lys Met Lys Tyr Asp Phe Ser CysGlu Leu Tyr 35 40 45 Arg Met Ser Thr Tyr Ser Thr Phe Pro Ala Gly Val ProVal Ser Glu 50 55 60 Arg Ser Leu Ala Arg Ala Gly Phe Tyr Tyr Thr Gly ValAsn Asp Lys 65 70 75 80 Val Lys Cys Phe Cys Cys Gly Leu Met Leu Asp AsnTrp Lys Leu Gly 85 90 95 Asp Ser Pro Ile Gln Lys His Lys Gln Leu Tyr ProSer Cys Ser Phe 100 105 110 Ile Gln Asn Leu Val Ser Ala Ser Leu Gly SerThr Ser Lys Asn Thr 115 120 125 Ser Pro Met Arg Asn Ser Phe Ala His SerLeu Ser Pro Thr Leu Glu 130 135 140 His Ser Ser Leu Phe Ser Gly Ser TyrSer Ser Leu Pro Pro Asn Pro 145 150 155 160 Leu Asn Ser Arg Ala Val GluAsp Ile Ser Ser Ser Arg Thr Asn Pro 165 170 175 Tyr Ser Tyr Ala Met SerThr Glu Glu Ala Arg Phe Leu Thr Tyr His 180 185 190 Met Trp Pro Leu ThrPhe Leu Ser Pro Ser Glu Leu Ala Arg Ala Gly 195 200 205 Phe Tyr Tyr IleGly Pro Gly Asp Arg Val Ala Cys Phe Ala Cys Gly 210 215 220 Gly Lys LeuSer Asn Trp Glu Pro Lys Asp Asp Ala Met Ser Glu His 225 230 235 240 ArgArg His Phe Pro Asn Cys Pro Phe Leu Glu Asn Ser Leu Glu Thr 245 250 255Leu Arg Phe Ser Ile Ser Asn Leu Ser Met Gln Thr His Ala Ala Arg 260 265270 Met Arg Thr Phe Met Tyr Trp Pro Ser Ser Val Pro Val Gln Pro Glu 275280 285 Gln Leu Ala Ser Ala Gly Phe Tyr Tyr Val Gly Arg Asn Asp Asp Val290 295 300 Lys Cys Phe Gly Cys Asp Gly Gly Leu Arg Cys Trp Glu Ser GlyAsp 305 310 315 320 Asp Pro Trp Val Glu His Ala Lys Trp Phe Pro Arg CysGlu Phe Leu 325 330 335 Ile Arg Met Lys Gly Gln Glu Phe Val Asp Glu IleGln Gly Arg Tyr 340 345 350 Pro His Leu Leu Glu Gln Leu Leu Ser Thr SerAsp Thr Thr Gly Glu 355 360 365 Glu Asn Ala Asp Pro Pro Ile Ile His PheGly Pro Gly Glu Ser Ser 370 375 380 Ser Glu Asp Ala Val Met Met Asn ThrPro Val Val Lys Ser Ala Leu 385 390 395 400 Glu Met Gly Phe Asn Arg AspLeu Val Lys Gln Thr Val Leu Ser Lys 405 410 415 Ile Leu Thr Thr Gly GluAsn Tyr Lys Thr Val Asn Asp Ile Val Ser 420 425 430 Ala Leu Leu Asn AlaGlu Asp Glu Lys Arg Glu Glu Glu Lys Glu Lys 435 440 445 Gln Ala Glu GluMet Ala Ser Asp Asp Leu Ser Leu Ile Arg Lys Asn 450 455 460 Arg Met AlaLeu Phe Gln Gln Leu Thr Cys Val Leu Pro Ile Leu Asp 465 470 475 480 AsnLeu Leu Lys Ala Asn Val Ile Asn Lys Gln Glu His Asp Ile Ile 485 490 495Lys Gln Lys Thr Gln Ile Pro Leu Gln Ala Arg Glu Leu Ile Asp Thr 500 505510 Ile Trp Val Lys Gly Asn Ala Ala Ala Asn Ile Phe Lys Asn Cys Leu 515520 525 Lys Glu Ile Asp Ser Thr Leu Tyr Lys Asn Leu Phe Val Asp Lys Asn530 535 540 Met Lys Tyr Ile Pro Thr Glu Asp Val Ser Gly Leu Ser Leu GluGlu 545 550 555 560 Gln Leu Arg Arg Leu Gln Glu Glu Arg Thr Cys Lys ValCys Met Asp 565 570 575 Lys Glu Val Ser Val Val Phe Ile Pro Cys Gly HisLeu Val Val Cys 580 585 590 Gln Glu Cys Ala Pro Ser Leu Arg Lys Cys ProIle Cys Arg Gly Ile 595 600 605 Ile Lys Gly Thr Val Arg Thr Phe Leu Ser610 615 9 2100 DNA Mus musculus 9 gacactctgc tgggcggcgg gccgccctcctccgggacct cccctcggga accgtcgccc 60 gcggcgctta gttaggactg gagtgcttggcgcgaaaagg tggacaagtc ctattttcca 120 gagaagatga cttttaacag ttttgaaggaactagaactt ttgtacttgc agacaccaat 180 aaggatgaag aatttgtaga agagtttaatagattaaaaa catttgctaa cttcccaagt 240 agtagtcctg tttcagcatc aacattggcgcgagctgggt ttctttatac cggtgaagga 300 gacaccgtgc aatgtttcag ttgtcatgcggcaatagata gatggcagta tggagactca 360 gctgttggaa gacacaggag aatatccccaaattgcagat ttatcaatgg tttttatttt 420 gaaaatggtg ctgcacagtc tacaaatcctggtatccaaa atggccagta caaatctgaa 480 aactgtgtgg gaaatagaaa tccttttgcccctgacaggc cacctgagac tcatgctgat 540 tatctcttga gaactggaca ggttgtagatatttcagaca ccatataccc gaggaaccct 600 gccatgtgta gtgaagaagc cagattgaagtcatttcaga actggccgga ctatgctcat 660 ttaaccccca gagagttagc tagtgctggcctctactaca caggggctga tgatcaagtg 720 caatgctttt gttgtggggg aaaactgaaaaattgggaac cctgtgatcg tgcctggtca 780 gaacacagga gacactttcc caattgcttttttgttttgg gccggaacgt taatgttcga 840 agtgaatctg gtgtgagttc tgataggaatttcccaaatt caacaaactc tccaagaaat 900 ccagccatgg cagaatatga agcacggatcgttacttttg gaacatggat atactcagtt 960 aacaaggagc agcttgcaag agctggattttatgctttag gtgaaggcga taaagtgaag 1020 tgcttccact gtggaggagg gctcacggattggaagccaa gtgaagaccc ctgggaccag 1080 catgctaagt gctacccagg gtgcaaatacctattggatg agaaggggca agaatatata 1140 aataatattc atttaaccca tccacttgaggaatctttgg gaagaactgc tgaaaaaaca 1200 ccaccgctaa ctaaaaaaat cgatgataccatcttccaga atcctatggt gcaagaagct 1260 atacgaatgg gatttagctt caaggaccttaagaaaacaa tggaagaaaa aatccaaaca 1320 tccgggagca gctatctatc acttgaggtcctgattgcag atcttgtgag tgctcagaaa 1380 gataatacgg aggatgagtc aagtcaaacttcattgcaga aagacattag tactgaagag 1440 cagctaaggc gcctacaaga ggagaagctttccaaaatct gtatggatag aaatattgct 1500 atcgtttttt ttccttgtgg acatctggccacttgtaaac agtgtgcaga agcagttgac 1560 aaatgtccca tgtgctacac cgtcattacgttcaaccaaa aaatttttat gtcttagtgg 1620 ggcaccacat gttatgttct tcttgctctaattgaatgtg taatgggagc gaactttaag 1680 taatcctgca tttgcattcc attagcatcctgctgtttcc aaatggagac caatgctaac 1740 agcactgttt ccgtctaaac attcaatttctggatctttc gagttatcag ctgtatcatt 1800 tagccagtgt tttactcgat tgaaaccttagacagagaag cattttatag cttttcacat 1860 gtatattggt agtacactga cttgatttctatatgtaagt gaattcatca cctgcatgtt 1920 tcatgccttt tgcataagct taacaaatggagtgttctgt ataagcatgg agatgtgatg 1980 gaatctgccc aatgacttta attggcttattgtaaacacg gaaagaactg ccccacgctg 2040 ctgggaggat aaagattgtt ttagatgctcacttctgtgt tttaggattc tgcccattta 2100 10 496 PRT Mus musculus 10 Met ThrPhe Asn Ser Phe Glu Gly Thr Arg Thr Phe Val Leu Ala Asp 1 5 10 15 ThrAsn Lys Asp Glu Glu Phe Val Glu Glu Phe Asn Arg Leu Lys Thr 20 25 30 PheAla Asn Phe Pro Ser Ser Ser Pro Val Ser Ala Ser Thr Leu Ala 35 40 45 ArgAla Gly Phe Leu Tyr Thr Gly Glu Gly Asp Thr Val Gln Cys Phe 50 55 60 SerCys His Ala Ala Ile Asp Arg Trp Gln Tyr Gly Asp Ser Ala Val 65 70 75 80Gly Arg His Arg Arg Ile Ser Pro Asn Cys Arg Phe Ile Asn Gly Phe 85 90 95Tyr Phe Glu Asn Gly Ala Ala Gln Ser Thr Asn Pro Gly Ile Gln Asn 100 105110 Gly Gln Tyr Lys Ser Glu Asn Cys Val Gly Asn Arg Asn Pro Phe Ala 115120 125 Pro Asp Arg Pro Pro Glu Thr His Ala Asp Tyr Leu Leu Arg Thr Gly130 135 140 Gln Val Val Asp Ile Ser Asp Thr Ile Tyr Pro Arg Asn Pro AlaMet 145 150 155 160 Cys Ser Glu Glu Ala Arg Leu Lys Ser Phe Gln Asn TrpPro Asp Tyr 165 170 175 Ala His Leu Thr Pro Arg Glu Leu Ala Ser Ala GlyLeu Tyr Tyr Thr 180 185 190 Gly Ala Asp Asp Gln Val Gln Cys Phe Cys CysGly Gly Lys Leu Lys 195 200 205 Asn Trp Glu Pro Cys Asp Arg Ala Trp SerGlu His Arg Arg His Phe 210 215 220 Pro Asn Cys Phe Phe Val Leu Gly ArgAsn Val Asn Val Arg Ser Glu 225 230 235 240 Ser Gly Val Ser Ser Asp ArgAsn Phe Pro Asn Ser Thr Asn Ser Pro 245 250 255 Arg Asn Pro Ala Met AlaGlu Tyr Glu Ala Arg Ile Val Thr Phe Gly 260 265 270 Thr Trp Ile Tyr SerVal Asn Lys Glu Gln Leu Ala Arg Ala Gly Phe 275 280 285 Tyr Ala Leu GlyGlu Gly Asp Lys Val Lys Cys Phe His Cys Gly Gly 290 295 300 Gly Leu ThrAsp Trp Lys Pro Ser Glu Asp Pro Trp Asp Gln His Ala 305 310 315 320 LysCys Tyr Pro Gly Cys Lys Tyr Leu Leu Asp Glu Lys Gly Gln Glu 325 330 335Tyr Ile Asn Asn Ile His Leu Thr His Pro Leu Glu Glu Ser Leu Gly 340 345350 Arg Thr Ala Glu Lys Thr Pro Pro Leu Thr Lys Lys Ile Asp Asp Thr 355360 365 Ile Phe Gln Asn Pro Met Val Gln Glu Ala Ile Arg Met Gly Phe Ser370 375 380 Phe Lys Asp Leu Lys Lys Thr Met Glu Glu Lys Ile Gln Thr SerGly 385 390 395 400 Ser Ser Tyr Leu Ser Leu Glu Val Leu Ile Ala Asp LeuVal Ser Ala 405 410 415 Gln Lys Asp Asn Thr Glu Asp Glu Ser Ser Gln ThrSer Leu Gln Lys 420 425 430 Asp Ile Ser Thr Glu Glu Gln Leu Arg Arg LeuGln Glu Glu Lys Leu 435 440 445 Ser Lys Ile Cys Met Asp Arg Asn Ile AlaIle Val Phe Phe Pro Cys 450 455 460 Gly His Leu Ala Thr Cys Lys Gln CysAla Glu Ala Val Asp Lys Cys 465 470 475 480 Pro Met Cys Tyr Thr Val IleThr Phe Asn Gln Lys Ile Phe Met Ser 485 490 495 11 67 PRT Orgyiapseudotsugata 11 Lys Ala Ala Arg Leu Gly Thr Tyr Thr Asn Trp Pro Val GlnPhe Leu 1 5 10 15 Glu Pro Ser Arg Met Ala Ala Ser Gly Phe Tyr Tyr LeuGly Arg Gly 20 25 30 Asp Glu Val Arg Cys Ala Phe Cys Lys Val Glu Ile ThrAsn Trp Val 35 40 45 Arg Gly Asp Asp Pro Glu Thr Asp His Lys Arg Trp AlaPro Gln Cys 50 55 60 Pro Phe Val 65 12 275 PRT Cydia pomonella 12 MetSer Asp Leu Arg Leu Glu Glu Val Arg Leu Asn Thr Phe Glu Lys 1 5 10 15Trp Pro Val Ser Phe Leu Ser Pro Glu Thr Met Ala Lys Asn Gly Phe 20 25 30Tyr Tyr Leu Gly Arg Ser Asp Glu Val Arg Cys Ala Phe Cys Lys Val 35 40 45Glu Ile Met Arg Trp Lys Glu Gly Glu Asp Pro Ala Ala Asp His Lys 50 55 60Lys Trp Ala Pro Gln Cys Pro Phe Val Lys Gly Ile Asp Val Cys Gly 65 70 7580 Ser Ile Val Thr Thr Asn Asn Ile Gln Asn Thr Thr Thr His Asp Thr 85 9095 Ile Ile Gly Pro Ala His Pro Lys Tyr Ala His Glu Ala Ala Arg Val 100105 110 Lys Ser Phe His Asn Trp Pro Arg Cys Met Lys Gln Arg Pro Glu Gln115 120 125 Met Ala Asp Ala Gly Phe Phe Tyr Thr Gly Tyr Gly Asp Asn ThrLys 130 135 140 Cys Phe Tyr Cys Asp Gly Gly Leu Lys Asp Trp Glu Pro GluAsp Val 145 150 155 160 Pro Trp Glu Gln His Val Arg Trp Phe Asp Arg CysAla Tyr Val Gln 165 170 175 Leu Val Lys Gly Arg Asp Tyr Val Gln Lys ValIle Thr Glu Ala Cys 180 185 190 Val Leu Pro Gly Glu Asn Thr Thr Val SerThr Ala Ala Pro Val Ser 195 200 205 Glu Pro Ile Pro Glu Thr Lys Ile GluLys Glu Pro Gln Val Glu Asp 210 215 220 Ser Lys Leu Cys Lys Ile Cys TyrVal Glu Glu Cys Ile Val Cys Phe 225 230 235 240 Val Pro Cys Gly His ValVal Ala Cys Ala Lys Cys Ala Leu Ser Val 245 250 255 Asp Lys Cys Pro MetCys Arg Lys Ile Val Thr Ser Val Leu Lys Val 260 265 270 Tyr Phe Ser 27513 498 PRT Drosophila melanogaster 13 Met Thr Glu Leu Gly Met Glu LeuGlu Ser Val Arg Leu Ala Thr Phe 1 5 10 15 Gly Glu Trp Pro Leu Asn AlaPro Val Ser Ala Glu Asp Leu Val Ala 20 25 30 Asn Gly Phe Phe Ala Thr GlyLys Trp Leu Glu Ala Glu Cys His Phe 35 40 45 Cys His Val Arg Ile Asp ArgTrp Glu Tyr Gly Asp Gln Val Ala Glu 50 55 60 Arg His Arg Arg Ser Ser ProIle Cys Ser Met Val Leu Ala Pro Asn 65 70 75 80 His Cys Gly Asn Val ProArg Ser Gln Glu Ser Asp Asn Glu Gly Asn 85 90 95 Ser Val Val Asp Ser ProGlu Ser Cys Ser Cys Pro Asp Leu Leu Leu 100 105 110 Glu Ala Asn Arg LeuVal Thr Phe Lys Asp Trp Pro Asn Pro Asn Ile 115 120 125 Thr Pro Gln AlaLeu Ala Lys Ala Gly Phe Tyr Tyr Leu Asn Arg Leu 130 135 140 Asp His ValLys Cys Val Trp Cys Asn Gly Val Ile Ala Lys Trp Glu 145 150 155 160 LysAsn Asp Asn Ala Phe Glu Glu His Lys Arg Phe Phe Pro Gln Cys 165 170 175Pro Arg Val Gln Met Gly Pro Leu Ile Glu Phe Ala Thr Gly Lys Asn 180 185190 Leu Asp Glu Leu Gly Ile Gln Pro Thr Thr Leu Pro Leu Arg Pro Lys 195200 205 Tyr Ala Cys Val Asp Ala Arg Leu Arg Thr Phe Thr Asp Trp Pro Ile210 215 220 Ser Asn Ile Gln Pro Ala Ser Ala Leu Ala Gln Ala Gly Leu TyrTyr 225 230 235 240 Gln Lys Ile Gly Asp Gln Val Arg Cys Phe His Cys AsnIle Gly Leu 245 250 255 Arg Ser Trp Gln Lys Glu Asp Glu Pro Trp Phe GluHis Ala Lys Trp 260 265 270 Ser Pro Lys Cys Gln Phe Val Leu Leu Ala LysGly Pro Ala Tyr Val 275 280 285 Ser Glu Val Leu Ala Thr Thr Ala Ala AsnAla Ser Ser Gln Pro Ala 290 295 300 Thr Ala Pro Ala Pro Thr Leu Gln AlaAsp Val Leu Met Asp Glu Ala 305 310 315 320 Pro Ala Lys Glu Ala Leu ThrLeu Gly Ile Asp Gly Gly Val Val Arg 325 330 335 Asn Ala Ile Gln Arg LysLeu Leu Ser Ser Gly Cys Ala Phe Ser Thr 340 345 350 Leu Asp Glu Leu LeuHis Asp Ile Phe Asp Asp Ala Gly Ala Gly Ala 355 360 365 Ala Leu Glu ValArg Glu Pro Pro Glu Pro Ser Ala Pro Phe Ile Glu 370 375 380 Pro Cys GlnAla Thr Thr Ser Lys Ala Ala Ser Val Pro Ile Pro Val 385 390 395 400 AlaAsp Ser Ile Pro Ala Lys Pro Gln Ala Ala Glu Ala Val Ser Asn 405 410 415Ile Ser Lys Ile Thr Asp Glu Ile Gln Lys Met Ser Val Ser Thr Pro 420 425430 Asn Gly Asn Leu Ser Leu Glu Glu Glu Asn Arg Gln Leu Lys Asp Ala 435440 445 Arg Leu Cys Lys Val Cys Leu Asp Glu Glu Val Gly Val Val Phe Leu450 455 460 Pro Cys Gly His Leu Ala Thr Cys Asn Gln Cys Ala Pro Ser ValAla 465 470 475 480 Asn Cys Pro Met Cys Arg Ala Asp Ile Lys Gly Phe ValArg Thr Phe 485 490 495 Leu Ser 14 67 PRT Cydia pomonella 14 Glu Glu ValArg Leu Asn Thr Phe Glu Lys Trp Pro Val Ser Phe Leu 1 5 10 15 Ser ProGlu Thr Met Ala Lys Asn Gly Phe Tyr Tyr Leu Gly Arg Ser 20 25 30 Asp GluVal Arg Cys Ala Phe Cys Lys Val Glu Ile Met Arg Trp Lys 35 40 45 Glu GlyGlu Asp Pro Ala Ala Asp His Lys Lys Trp Ala Pro Gln Cys 50 55 60 Pro PheVal 65 15 67 PRT Drosophila melanogaster 15 Glu Ala Asn Arg Leu Val ThrPhe Lys Asp Trp Pro Asn Pro Asn Ile 1 5 10 15 Thr Pro Gln Ala Leu AlaLys Ala Gly Phe Tyr Tyr Leu Asn Arg Leu 20 25 30 Asp His Val Lys Cys ValTrp Cys Asn Gly Val Ile Ala Lys Trp Glu 35 40 45 Lys Asn Asp Asn Ala PheGlu Glu His Lys Arg Phe Phe Pro Gln Cys 50 55 60 Pro Arg Val 65 16 68PRT Mus musculus 16 Glu Phe Asn Arg Leu Lys Thr Phe Ala Asn Phe Pro SerSer Ser Pro 1 5 10 15 Val Ser Ala Ser Thr Leu Ala Arg Ala Gly Phe LeuTyr Thr Gly Glu 20 25 30 Gly Asp Thr Val Gln Cys Phe Ser Cys His Ala AlaIle Asp Arg Trp 35 40 45 Gln Tyr Gly Asp Ser Ala Val Gly Arg His Arg ArgIle Ser Pro Asn 50 55 60 Cys Arg Phe Ile 65 17 68 PRT Homo sapiens 17Glu Phe Asn Arg Leu Lys Thr Phe Ala Asn Phe Pro Ser Gly Ser Pro 1 5 1015 Val Ser Ala Ser Thr Leu Ala Arg Ala Gly Phe Leu Tyr Thr Gly Glu 20 2530 Gly Asp Thr Val Arg Cys Phe Ser Cys His Ala Ala Val Asp Arg Trp 35 4045 Gln Tyr Gly Asp Ser Ala Val Gly Arg His Arg Lys Val Ser Pro Asn 50 5560 Cys Arg Phe Ile 65 18 68 PRT Homo sapiens 18 Glu Leu Tyr Arg Met SerThr Tyr Ser Thr Phe Pro Ala Gly Val Pro 1 5 10 15 Val Ser Glu Arg SerLeu Ala Arg Ala Gly Phe Tyr Tyr Thr Gly Val 20 25 30 Asn Asp Lys Val LysCys Phe Cys Cys Gly Leu Met Leu Asp Asn Trp 35 40 45 Lys Arg Gly Asp SerPro Thr Glu Lys His Lys Lys Leu Tyr Pro Ser 50 55 60 Cys Arg Phe Val 6519 68 PRT Homo sapiens 19 Glu Leu Tyr Arg Met Ser Thr Tyr Ser Thr PhePro Ala Gly Val Pro 1 5 10 15 Val Ser Glu Arg Ser Leu Ala Arg Ala GlyPhe Tyr Tyr Thr Gly Val 20 25 30 Asn Asp Lys Val Lys Cys Phe Cys Cys GlyLeu Met Leu Asp Asn Trp 35 40 45 Lys Leu Gly Asp Ser Pro Ile Gln Lys HisLys Gln Leu Tyr Pro Ser 50 55 60 Cys Ser Phe Ile 65 20 68 PRT Musmusculus 20 Glu Glu Ala Arg Leu Lys Ser Phe Gln Asn Trp Pro Asp Tyr AlaHis 1 5 10 15 Leu Thr Pro Arg Glu Leu Ala Ser Ala Gly Leu Tyr Tyr ThrGly Ala 20 25 30 Asp Asp Gln Val Gln Cys Phe Cys Cys Gly Gly Lys Leu LysAsn Trp 35 40 45 Glu Pro Cys Asp Arg Ala Trp Ser Glu His Arg Arg His PhePro Asn 50 55 60 Cys Phe Phe Val 65 21 68 PRT Homo sapiens 21 Glu GluAla Arg Leu Lys Ser Phe Gln Asn Trp Pro Asp Tyr Ala His 1 5 10 15 LeuThr Pro Arg Glu Leu Ala Ser Ala Gly Leu Tyr Tyr Thr Gly Ile 20 25 30 GlyAsp Gln Val Gln Cys Phe Cys Cys Gly Gly Lys Leu Lys Asn Trp 35 40 45 GluPro Cys Asp Arg Ala Trp Ser Glu His Arg Arg His Phe Pro Asn 50 55 60 CysPhe Phe Val 65 22 67 PRT Homo sapiens 22 Glu Asn Ala Arg Leu Leu Thr PheGln Thr Trp Pro Leu Thr Phe Leu 1 5 10 15 Ser Pro Thr Asp Leu Ala ArgAla Gly Phe Tyr Tyr Ile Gly Pro Gly 20 25 30 Asp Arg Val Ala Cys Phe AlaCys Gly Gly Lys Leu Ser Asn Trp Glu 35 40 45 Pro Lys Asp Asn Ala Met SerGlu His Leu Arg His Phe Pro Lys Cys 50 55 60 Pro Phe Ile 65 23 67 PRTHomo sapiens 23 Glu Glu Ala Arg Phe Leu Thr Tyr His Met Trp Pro Leu ThrPhe Leu 1 5 10 15 Ser Pro Ser Glu Leu Ala Arg Ala Gly Phe Tyr Tyr IleGly Pro Gly 20 25 30 Asp Arg Val Ala Cys Phe Ala Cys Gly Gly Lys Leu SerAsn Trp Glu 35 40 45 Pro Lys Asp Asp Ala Met Ser Glu His Arg Arg His PhePro Asn Cys 50 55 60 Pro Phe Leu 65 24 66 PRT Mus musculus 24 Tyr GluAla Arg Ile Val Thr Phe Gly Thr Trp Ile Tyr Ser Val Asn 1 5 10 15 LysGlu Gln Leu Ala Arg Ala Gly Phe Tyr Ala Leu Gly Glu Gly Asp 20 25 30 LysVal Lys Cys Phe His Cys Gly Gly Gly Leu Thr Asp Trp Lys Pro 35 40 45 SerGlu Asp Pro Trp Asp Gln His Ala Lys Cys Tyr Pro Gly Cys Lys 50 55 60 TyrLeu 65 25 66 PRT Homo sapiens 25 Tyr Glu Ala Arg Ile Phe Thr Phe Gly ThrTrp Ile Tyr Ser Val Asn 1 5 10 15 Lys Glu Gln Leu Ala Arg Ala Gly PheTyr Ala Leu Gly Glu Gly Asp 20 25 30 Lys Val Lys Cys Phe His Cys Gly GlyGly Leu Thr Asp Trp Lys Pro 35 40 45 Ser Glu Asp Pro Trp Glu Gln His AlaLys Trp Tyr Pro Gly Cys Lys 50 55 60 Tyr Leu 65 26 68 PRT Homo sapiens26 His Ala Ala Arg Phe Lys Thr Phe Phe Asn Trp Pro Ser Ser Val Leu 1 510 15 Val Asn Pro Glu Gln Leu Ala Ser Ala Gly Phe Tyr Tyr Val Gly Asn 2025 30 Ser Asp Asp Val Lys Cys Phe Cys Cys Asp Gly Gly Leu Arg Cys Trp 3540 45 Glu Ser Gly Asp Asp Pro Trp Val Gln His Ala Lys Trp Phe Pro Arg 5055 60 Cys Glu Tyr Leu 65 27 68 PRT Homo sapiens 27 His Ala Ala Arg MetArg Thr Phe Met Tyr Trp Pro Ser Ser Val Pro 1 5 10 15 Val Gln Pro GluGln Leu Ala Ser Ala Gly Phe Tyr Tyr Val Gly Arg 20 25 30 Asn Asp Asp ValLys Cys Phe Gly Cys Asp Gly Gly Leu Arg Cys Trp 35 40 45 Glu Ser Gly AspAsp Pro Trp Val Glu His Ala Lys Trp Phe Pro Arg 50 55 60 Cys Glu Phe Leu65 28 68 PRT Orgyia pseudotsugata 28 Glu Ala Ala Arg Leu Arg Thr Phe AlaGlu Trp Pro Arg Gly Leu Lys 1 5 10 15 Gln Arg Pro Glu Glu Leu Ala GluAla Gly Phe Phe Tyr Thr Gly Gln 20 25 30 Gly Asp Lys Thr Arg Cys Phe CysCys Asp Gly Gly Leu Lys Asp Trp 35 40 45 Glu Pro Asp Asp Ala Pro Trp GlnGln His Ala Arg Trp Tyr Asp Arg 50 55 60 Cys Glu Tyr Val 65 29 68 PRTCydia pomonella 29 Glu Ala Ala Arg Val Lys Ser Phe His Asn Trp Pro ArgCys Met Lys 1 5 10 15 Gln Arg Pro Glu Gln Met Ala Asp Ala Gly Phe PheTyr Thr Gly Tyr 20 25 30 Gly Asp Asn Thr Lys Cys Phe Tyr Cys Asp Gly GlyLeu Lys Asp Trp 35 40 45 Glu Pro Glu Asp Val Pro Trp Glu Gln His Val ArgTrp Phe Asp Arg 50 55 60 Cys Ala Tyr Val 65 30 68 PRT Drosophilamelanogaster 30 Val Asp Ala Arg Leu Arg Thr Phe Thr Asp Trp Pro Ile SerAsn Ile 1 5 10 15 Gln Pro Ala Ser Ala Leu Ala Gln Ala Gly Leu Tyr TyrGln Lys Ile 20 25 30 Gly Asp Gln Val Arg Cys Phe His Cys Asn Ile Gly LeuArg Ser Trp 35 40 45 Gln Lys Glu Asp Glu Pro Trp Phe Glu His Ala Lys TrpSer Pro Lys 50 55 60 Cys Gln Phe Val 65 31 66 PRT Drosophilamelanogaster 31 Glu Ser Val Arg Leu Ala Thr Phe Gly Glu Trp Pro Leu AsnAla Pro 1 5 10 15 Val Ser Ala Glu Asp Leu Val Ala Asn Gly Phe Phe GlyThr Trp Met 20 25 30 Glu Ala Glu Cys Asp Phe Cys His Val Arg Ile Asp ArgTrp Glu Tyr 35 40 45 Gly Asp Leu Val Ala Glu Arg His Arg Arg Ser Ser ProIle Cys Ser 50 55 60 Met Val 65 32 46 PRT Homo sapiens 32 Glu Gln LeuArg Arg Leu Gln Glu Glu Arg Thr Cys Lys Val Cys Met 1 5 10 15 Asp LysGlu Val Ser Val Val Phe Ile Pro Cys Gly His Leu Val Val 20 25 30 Cys GlnGlu Cys Ala Pro Ser Leu Arg Lys Cys Pro Ile Cys 35 40 45 33 46 PRT Homosapiens 33 Glu Gln Leu Arg Arg Leu Pro Glu Glu Arg Thr Cys Lys Val CysMet 1 5 10 15 Asp Lys Glu Val Ser Ile Val Phe Ile Pro Cys Gly His LeuVal Val 20 25 30 Cys Lys Asp Cys Ala Pro Ser Leu Arg Lys Cys Pro Ile Cys35 40 45 34 46 PRT Mus musculus 34 Glu Gln Leu Arg Arg Leu Gln Glu GluLys Leu Ser Lys Ile Cys Met 1 5 10 15 Asp Arg Asn Ile Ala Ile Val PhePhe Pro Cys Gly His Leu Ala Thr 20 25 30 Cys Lys Gln Cys Ala Glu Ala ValAsp Lys Cys Pro Met Cys 35 40 45 35 46 PRT Homo sapiens 35 Glu Gln LeuArg Arg Leu Gln Glu Glu Lys Leu Cys Lys Ile Cys Met 1 5 10 15 Asp ArgAsn Ile Ala Ile Val Phe Val Pro Cys Gly His Leu Val Thr 20 25 30 Cys LysGln Cys Ala Glu Ala Val Asp Lys Cys Pro Met Cys 35 40 45 36 46 PRTDrosophila melanogaster 36 Glu Glu Asn Arg Gln Leu Lys Asp Ala Arg LeuCys Lys Val Cys Leu 1 5 10 15 Asp Glu Glu Val Gly Val Val Phe Leu ProCys Gly His Leu Ala Thr 20 25 30 Cys Asn Gln Cys Ala Pro Ser Val Ala AsnCys Pro Met Cys 35 40 45 37 46 PRT Cydia pomonella 37 Glu Lys Glu ProGln Val Glu Asp Ser Lys Leu Cys Lys Ile Cys Tyr 1 5 10 15 Val Glu GluCys Ile Val Cys Phe Val Pro Cys Gly His Val Val Ala 20 25 30 Cys Ala LysCys Ala Leu Ser Val Asp Lys Cys Pro Met Cys 35 40 45 38 46 PRT Orgyiapseudotsugata 38 Ala Val Glu Ala Glu Val Ala Asp Asp Arg Leu Cys Lys IleCys Leu 1 5 10 15 Gly Ala Glu Lys Thr Val Cys Phe Val Pro Cys Gly HisVal Val Ala 20 25 30 Cys Gly Lys Cys Ala Ala Gly Val Thr Thr Cys Pro ValCys 35 40 45 39 2474 DNA Mus musculus 39 gaattccggg agacctacacccccggagat cagaggtcat tgctggcgtt cagagcctag 60 gaagtgggct gcggtatcagcctagcagta aaaccgacca gaagccatgc acaaaactac 120 atccccagag aaagacttgtcccttcccct ccctgtcatc tcaccatgaa catggttcaa 180 gacagcgcct ttctagccaagctgatgaag agtgctgaca cctttgagtt gaagtatgac 240 ttttcctgtg agctgtaccgattgtccacg tattcagctt ttcccagggg agttcctgtg 300 tcagaaagga gtctggctcgtgctggcttt tactacactg gtgccaatga caaggtcaag 360 tgcttctgct gtggcctgatgctagacaac tggaaacaag gggacagtcc catggagaag 420 cacagaaagt tgtaccccagctgcaacttt gtacagactt tgaatccagc caacagtctg 480 gaagctagtc ctcggccttctcttccttcc acggcgatga gcaccatgcc tttgagcttt 540 gcaagttctg agaatactggctatttcagt ggctcttact cgagctttcc ctcagaccct 600 gtgaacttcc gagcaaatcaagattgtcct gctttgagca caagtcccta ccactttgca 660 atgaacacag agaaggccagattactcacc tatgaaacat ggccattgtc ttttctgtca 720 ccagcaaagc tggccaaagcaggcttctac tacataggac ctggagatag agtggcctgc 780 tttgcgtgcg atgggaaactgagcaactgg gaacgtaagg atgatgctat gtcagagcac 840 cagaggcatt tccccagctgtccgttctta aaagacttgg gtcagtctgc ttcgagatac 900 actgtctcta acctgagcatgcagacacac gcagcccgta ttagaacatt ctctaactgg 960 ccttctagtg cactagttcattcccaggaa cttgcaagtg cgggctttta ttatacagga 1020 cacagtgatg atgtcaagtgtttatgctgt gatggtgggc tgaggtgctg ggaatctgga 1080 gatgacccct gggtggaacatgccaagtgg tttccaaggt gtgagtactt gctcagaatc 1140 aaaggccaag aatttgtcagccaagttcaa gctggctatc ctcatctact tgagcagcta 1200 ttatctacgt cagactccccagaagatgag aatgcagacg cagcaatcgt gcattttggc 1260 cctggagaaa gttcggaagatgtcgtcatg atgagcacgc ctgtggttaa agcagccttg 1320 gaaatgggct tcagtaggagcctggtgaga cagacggttc agtggcagat cctggccact 1380 ggtgagaact acaggaccgtcagtgacctc gttataggct tactcgatgc agaagacgag 1440 atgagagagg agcagatggagcaggcggcc gaggaggagg agtcagatga tctagcacta 1500 atccggaaga acaaaatggtgcttttccaa catttgacgt gtgtgacacc aatgctgtat 1560 tgcctcctaa gtgcaagggccatcactgaa caggagtgca atgctgtgaa acagaaacca 1620 cacaccttac aagcaagcacactgattgat actgtgttag caaaaggaaa cactgcagca 1680 acctcattca gaaactcccttcgggaaatt gaccctgcgt tatacagaga tatatttgtg 1740 caacaggaca ttaggagtcttcccacagat gacattgcag ctctaccaat ggaagaacag 1800 ttgcggcccc tcccggaggacagaatgtgt aaagtgtgta tggaccgaga ggtatccatc 1860 gtgttcattc cctgtggccatctggtcgtg tgcaaagact gcgctccctc tctgaggaag 1920 tgtcccatct gtagagggaccatcaagggc acagtgcgca catttctctc ctgaacaaga 1980 ctaatggtcc atggctgcaacttcagccag gaggaagttc actgtcactc ccagttccat 2040 tcggaacttg aggccagcctggatagcacg agacaccgcc aaacacacaa atataaacat 2100 gaaaaacttt tgtctgaagtcaagaatgaa tgaattactt atataataat tttaattggt 2160 ttccttaaaa gtgctatttgttcccaactc agaaaattgt tttctgtaaa catatttaca 2220 tactacctgc atctaaagtattcatatatt catatattca gatgtcatga gagagggttt 2280 tgttcttgtt cctgaaaagctggtttatca tctgatcagc atatactgcg caacgggcag 2340 ggctagaatc catgaaccaagctgcaaaga tctcacgcta aataaggcgg aaagatttgg 2400 agaaacgaaa ggaaattctttcctgtccaa tgtatactct tcagactaat gacctcttcc 2460 tatcaagcct tcta 2474 40602 PRT Mus musculus 40 Met Asn Met Val Gln Asp Ser Ala Phe Leu Ala LysLeu Met Lys Ser 1 5 10 15 Ala Asp Thr Phe Glu Leu Lys Tyr Asp Phe SerCys Glu Leu Tyr Arg 20 25 30 Leu Ser Thr Tyr Ser Ala Phe Pro Arg Gly ValPro Val Ser Glu Arg 35 40 45 Ser Leu Ala Arg Ala Gly Phe Tyr Tyr Thr GlyAla Asn Asp Lys Val 50 55 60 Lys Cys Phe Cys Cys Gly Leu Met Leu Asp AsnTrp Lys Gln Gly Asp 65 70 75 80 Ser Pro Met Glu Lys His Arg Lys Leu TyrPro Ser Cys Asn Phe Val 85 90 95 Gln Thr Leu Asn Pro Ala Asn Ser Leu GluAla Ser Pro Arg Pro Ser 100 105 110 Leu Pro Ser Thr Ala Met Ser Thr MetPro Leu Ser Phe Ala Ser Ser 115 120 125 Glu Asn Thr Gly Tyr Phe Ser GlySer Tyr Ser Ser Phe Pro Ser Asp 130 135 140 Pro Val Asn Phe Arg Ala AsnGln Asp Cys Pro Ala Leu Ser Thr Ser 145 150 155 160 Pro Tyr His Phe AlaMet Asn Thr Glu Lys Ala Arg Leu Leu Thr Tyr 165 170 175 Glu Thr Trp ProLeu Ser Phe Leu Ser Pro Ala Lys Leu Ala Lys Ala 180 185 190 Gly Phe TyrTyr Ile Gly Pro Gly Asp Arg Val Ala Cys Phe Ala Cys 195 200 205 Asp GlyLys Leu Ser Asn Trp Glu Arg Lys Asp Asp Ala Met Ser Glu 210 215 220 HisGln Arg His Phe Pro Ser Cys Pro Phe Leu Lys Asp Leu Gly Gln 225 230 235240 Ser Ala Ser Arg Tyr Thr Val Ser Asn Leu Ser Met Gln Thr His Ala 245250 255 Ala Arg Ile Arg Thr Phe Ser Asn Trp Pro Ser Ser Ala Leu Val His260 265 270 Ser Gln Glu Leu Ala Ser Ala Gly Phe Tyr Tyr Thr Gly His SerAsp 275 280 285 Asp Val Lys Cys Leu Cys Cys Asp Gly Gly Leu Arg Cys TrpGlu Ser 290 295 300 Gly Asp Asp Pro Trp Val Glu His Ala Lys Trp Phe ProArg Cys Glu 305 310 315 320 Tyr Leu Leu Arg Ile Lys Gly Gln Glu Phe ValSer Gln Val Gln Ala 325 330 335 Gly Tyr Pro His Leu Leu Glu Gln Leu LeuSer Thr Ser Asp Ser Pro 340 345 350 Glu Asp Glu Asn Ala Asp Ala Ala IleVal His Phe Gly Pro Gly Glu 355 360 365 Ser Ser Glu Asp Val Val Met MetSer Thr Pro Val Val Lys Ala Ala 370 375 380 Leu Glu Met Gly Phe Ser ArgSer Leu Val Arg Gln Thr Val Gln Trp 385 390 395 400 Gln Ile Leu Ala ThrGly Glu Asn Tyr Arg Thr Val Ser Asp Leu Val 405 410 415 Ile Gly Leu LeuAsp Ala Glu Asp Glu Met Arg Glu Glu Gln Met Glu 420 425 430 Gln Ala AlaGlu Glu Glu Glu Ser Asp Asp Leu Ala Leu Ile Arg Lys 435 440 445 Asn LysMet Val Leu Phe Gln His Leu Thr Cys Val Thr Pro Met Leu 450 455 460 TyrCys Leu Leu Ser Ala Arg Ala Ile Thr Glu Gln Glu Cys Asn Ala 465 470 475480 Val Lys Gln Lys Pro His Thr Leu Gln Ala Ser Thr Leu Ile Asp Thr 485490 495 Val Leu Ala Lys Gly Asn Thr Ala Ala Thr Ser Phe Arg Asn Ser Leu500 505 510 Arg Glu Ile Asp Pro Ala Leu Tyr Arg Asp Ile Phe Val Gln GlnAsp 515 520 525 Ile Arg Ser Leu Pro Thr Asp Asp Ile Ala Ala Leu Pro MetGlu Glu 530 535 540 Gln Leu Arg Pro Leu Pro Glu Asp Arg Met Cys Lys ValCys Met Asp 545 550 555 560 Arg Glu Val Ser Ile Val Phe Ile Pro Cys GlyHis Leu Val Val Cys 565 570 575 Lys Asp Cys Ala Pro Ser Leu Arg Lys CysPro Ile Cys Arg Gly Thr 580 585 590 Ile Lys Gly Thr Val Arg Thr Phe LeuSer 595 600 41 2416 DNA Mus musculus 41 ctgtggtgga gatctattgt ccaagtggtgagaaacttca tctggaagtt taagcggtca 60 gaaatactat tactactcat ggacaaaactgtctcccaga gactcgccca aggtacctta 120 cacccaaaaa cttaaacgta taatggagaagagcacaatc ttgtcaaatt ggacaaagga 180 gagcgaagaa aaaatgaagt ttgacttttcgtgtgaactc taccgaatgt ctacatattc 240 agcttttccc aggggagttc ctgtctcagagaggagtctg gctcgtgctg gcttttatta 300 tacaggtgtg aatgacaaag tcaagtgcttctgctgtggc ctgatgttgg ataactggaa 360 acaaggggac agtcctgttg aaaagcacagacagttctat cccagctgca gctttgtaca 420 gactctgctt tcagccagtc tgcagtctccatctaagaat atgtctcctg tgaaaagtag 480 atttgcacat tcgtcacctc tggaacgaggtggcattcac tccaacctgt gctctagccc 540 tcttaattct agagcagtgg aagacttctcatcaaggatg gatccctgca gctatgccat 600 gagtacagaa gaggccagat ttcttacttacagtatgtgg cctttaagtt ttctgtcacc 660 agcagagctg gccagagctg gcttctattacatagggcct ggagacaggg tggcctgttt 720 tgcctgtggt gggaaactga gcaactgggaaccaaaggat tatgctatgt cagagcaccg 780 cagacatttt ccccactgtc catttctggaaaatacttca gaaacacaga ggtttagtat 840 atcaaatcta agtatgcaga cacactctgctcgattgagg acatttctgt actggccacc 900 tagtgttcct gttcagcccg agcagcttgcaagtgctgga ttctattacg tggatcgcaa 960 tgatgatgtc aagtgccttt gttgtgatggtggcttgaga tgttgggaac ctggagatga 1020 cccctggata gaacacgcca aatggtttccaaggtgtgag ttcttgatac ggatgaaggg 1080 tcaggagttt gttgatgaga ttcaagctagatatcctcat cttcttgagc agctgttgtc 1140 cacttcagac accccaggag aagaaaatgctgaccctaca gagacagtgg tgcattttgg 1200 ccctggagaa agttcgaaag atgtcgtcatgatgagcacg cctgtggtta aagcagcctt 1260 ggaaatgggc ttcagtagga gcctggtgagacagacggtt cagcggcaga tcctggccac 1320 tggtgagaac tacaggaccg tcaatgatattgtctcagta cttttgaatg ctgaagatga 1380 gagaagagaa gaggagaagg aaagacagactgaagagatg gcatcaggtg acttatcact 1440 gattcggaag aatagaatgg ccctctttcaacagttgaca catgtccttc ctatcctgga 1500 taatcttctt gaggccagtg taattacaaaacaggaacat gatattatta gacagaaaac 1560 acagataccc ttacaagcaa gagagcttattgacaccgtt ttagtcaagg gaaatgctgc 1620 agccaacatc ttcaaaaact ctctgaagggaattgactcc acgttatatg aaaacttatt 1680 tgtggaaaag aatatgaagt atattccaacagaagacgtt tcaggcttgt cattggaaga 1740 gcagttgcgg agattacaag aagaacgaacttgcaaagtg tgtatggaca gagaggtttc 1800 tattgtgttc attccgtgtg gtcatctagtagtctgccag gaatgtgccc cttctctaag 1860 gaagtgcccc atctgcaggg ggacaatcaaggggactgtg cgcacatttc tctcatgagt 1920 gaagaatggt ctgaaagtat tgttggacatcagaagctgt cagaacaaag aatgaactac 1980 tgatttcagc tcttcagcag gacattctactctctttcaa gattagtaat cttgctttat 2040 gaagggtagc attgtatatt taagcttagtctgttgcaag ggaaggtcta tgctgttgag 2100 ctacaggact gtgtctgttc cagagcaggagttgggatgc ttgctgtatg tccttcagga 2160 cttcttggga tttgggaatt tggggaaagctttggaatcc agtgatgtgg agctcagaaa 2220 tcctggaacc agtgactctg gtactcagtagatagggtac cctgtacttc ttggtgcttt 2280 tccagtctgg gaaataagga ggaatctgctgctggtaaaa atttgctgga tgtgagaaat 2340 agatgaaagt gtttcgggtg ggggcgtgcatcagtgtagt gtgtgcaggg atgtatgcag 2400 gccaaacact gtgtag 2416 42 591 PRTMus musculus 42 Met Glu Lys Ser Thr Ile Leu Ser Asn Trp Thr Lys Glu SerGlu Glu 1 5 10 15 Lys Met Lys Phe Asp Phe Ser Cys Glu Leu Tyr Arg MetSer Thr Tyr 20 25 30 Ser Ala Phe Pro Arg Gly Val Pro Val Ser Glu Arg SerLeu Ala Arg 35 40 45 Ala Gly Phe Tyr Tyr Thr Gly Val Asn Asp Lys Val LysCys Phe Cys 50 55 60 Cys Gly Leu Met Leu Asp Asn Trp Lys Gln Gly Asp SerPro Val Glu 65 70 75 80 Lys His Arg Gln Phe Tyr Pro Ser Cys Ser Phe ValGln Thr Leu Leu 85 90 95 Ser Ala Ser Leu Gln Ser Pro Ser Lys Asn Met SerPro Val Lys Ser 100 105 110 Arg Phe Ala His Ser Ser Pro Leu Glu Arg GlyGly Ile His Ser Asn 115 120 125 Leu Cys Ser Ser Pro Leu Asn Ser Arg AlaVal Glu Asp Phe Ser Ser 130 135 140 Arg Met Asp Pro Cys Ser Tyr Ala MetSer Thr Glu Glu Ala Arg Phe 145 150 155 160 Leu Thr Tyr Ser Met Trp ProLeu Ser Phe Leu Ser Pro Ala Glu Leu 165 170 175 Ala Arg Ala Gly Phe TyrTyr Ile Gly Pro Gly Asp Arg Val Ala Cys 180 185 190 Phe Ala Cys Gly GlyLys Leu Ser Asn Trp Glu Pro Lys Asp Tyr Ala 195 200 205 Met Ser Glu HisArg Arg His Phe Pro His Cys Pro Phe Leu Glu Asn 210 215 220 Thr Ser GluThr Gln Arg Phe Ser Ile Ser Asn Leu Ser Met Gln Thr 225 230 235 240 HisSer Ala Arg Leu Arg Thr Phe Leu Tyr Trp Pro Pro Ser Val Pro 245 250 255Val Gln Pro Glu Gln Leu Ala Ser Ala Gly Phe Tyr Tyr Val Asp Arg 260 265270 Asn Asp Asp Val Lys Cys Leu Cys Cys Asp Gly Gly Leu Arg Cys Trp 275280 285 Glu Pro Gly Asp Asp Pro Trp Ile Glu His Ala Lys Trp Phe Pro Arg290 295 300 Cys Glu Phe Leu Ile Arg Met Lys Gly Gln Glu Phe Val Asp GluIle 305 310 315 320 Gln Ala Arg Tyr Pro His Leu Leu Glu Gln Leu Leu SerThr Ser Asp 325 330 335 Thr Pro Gly Glu Glu Asn Ala Asp Pro Thr Glu ThrVal Val His Phe 340 345 350 Gly Pro Gly Glu Ser Ser Lys Asp Val Val MetMet Ser Thr Pro Val 355 360 365 Val Lys Ala Ala Leu Glu Met Gly Phe SerArg Ser Leu Val Arg Gln 370 375 380 Thr Val Gln Arg Gln Ile Leu Ala ThrGly Glu Asn Tyr Arg Thr Val 385 390 395 400 Asn Asp Ile Val Ser Val LeuLeu Asn Ala Glu Asp Glu Arg Arg Glu 405 410 415 Glu Glu Lys Glu Arg GlnThr Glu Glu Met Ala Ser Gly Asp Leu Ser 420 425 430 Leu Ile Arg Lys AsnArg Met Ala Leu Phe Gln Gln Leu Thr His Val 435 440 445 Leu Pro Ile LeuAsp Asn Leu Leu Glu Ala Ser Val Ile Thr Lys Gln 450 455 460 Glu His AspIle Ile Arg Gln Lys Thr Gln Ile Pro Leu Gln Ala Arg 465 470 475 480 GluLeu Ile Asp Thr Val Leu Val Lys Gly Asn Ala Ala Ala Asn Ile 485 490 495Phe Lys Asn Ser Leu Lys Gly Ile Asp Ser Thr Leu Tyr Glu Asn Leu 500 505510 Phe Val Glu Lys Asn Met Lys Tyr Ile Pro Thr Glu Asp Val Ser Gly 515520 525 Leu Ser Leu Glu Glu Gln Leu Arg Arg Leu Gln Glu Glu Arg Thr Cys530 535 540 Lys Val Cys Met Asp Arg Glu Val Ser Ile Val Phe Ile Pro CysGly 545 550 555 560 His Leu Val Val Cys Gln Glu Cys Ala Pro Ser Leu ArgLys Cys Pro 565 570 575 Ile Cys Arg Gly Thr Ile Lys Gly Thr Val Arg ThrPhe Leu Ser 580 585 590 43 11 PRT Artificial Sequence Synthetic based onviral sequence 43 Met Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 4421 DNA Artificial Sequence Synthetic primer based on Homo sapiens 44agtgcgggtt tttattatgt g 21 45 25 DNA Artificial Sequence Syntheticprimer based on Homo sapiens 45 agatgaccac aaggaataaa cacta 25

What is claimed is:
 1. A substantially pure polypeptide, wherein saidpolypeptide has at least 95% amino acid sequence identity to any one ofSEQ ID NOS: 4, 6, 8, 10, or 40, and comprises a domain having a sequenceselected from the group consisting of amino acids 26-93, 163-230, or265-330 of SEQ ID NO: 4, amino acids 29-96, 169-235, or 255-322 of SEQID NO: 6, amino acids 46-113, 184-250, or 269-336 of SEQ ID NO: 8, aminoacids 26-93, 163-230, or 264-329 of SEQ ID NO: 10, or amino acids 29-96,169-235, or 255-322 of SEQ ID NO: 40, wherein said polypeptide inhibitsapoptosis.
 2. The polypeptide of claim 1, wherein said polypeptide is ahuman polypeptide.
 3. The polypeptide of claim 1, wherein saidpolypeptide is M-XIAP (SEQ ID NO: 10), or M-HIAP-1 (SEQ ID NO: 40). 4.The polypeptide of claim 2, wherein said polypeptide is XIAP (SEQ ID NO:4), HIAP-1 (SEQ ID NO: 6), or HIAP-2 (SEQ ID NO: 8).
 5. The polypeptideof claim 4, wherein said polypeptide is XIAP (SEQ ID NO: 4).
 6. Thepolypeptide of claim wherein 3, said polypeptide is M-XIAP (SEQ ID NO:10).